Methods and systems for processing polynucleotides

ABSTRACT

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/052,486, filed Aug. 1, 2018, which is a continuation-in-part of U.S.application Ser. No. 16/000,803, filed Jun. 5, 2018, which is acontinuation of U.S. application Ser. No. 15/850,241, filed Dec. 21,2017, which is a continuation of U.S. patent application Ser. No.15/588,519, filed May 5, 2017, now U.S. Pat. No. 9,856,530, which is acontinuation of U.S. patent application Ser. No. 15/376,582, filed Dec.12, 2016, now U.S. Pat. No. 9,701,998, which is a continuation-in-partof U.S. patent application Ser. No. 14/104,650, filed on Dec. 12, 2013,now U.S. Pat. No. 9,567,631, which claims priority to U.S. ProvisionalApplication No. 61/737,374, filed on Dec. 14, 2012; U.S. patentapplication Ser. No. 15/376,582 is also a continuation-in-part of U.S.patent application Ser. No. 14/250,701, filed on Apr. 11, 2014, which isa continuation of U.S. patent application Ser. No. 14/175,973, filed onFeb. 7, 2014, now U.S. Pat. No. 9,388,465, which claims priority to U.S.Provisional Application No. 61/844,804, filed on Jul. 10, 2013, U.S.Provisional Application No. 61/840,403, filed on Jun. 27, 2013, U.S.Provisional Application No. 61/800,223, filed on Mar. 15, 2013, and U.S.Provisional Application No. 61/762,435, filed on Feb. 8, 2013, each ofwhich is entirely incorporated herein by reference for all purposes.U.S. patent application Ser. No. 16/052,486, filed Aug. 1, 2018, is alsoa continuation-in-part of U.S. application Ser. No. 14/316,447, filed onJun. 26, 2014, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/966,150, filed on Aug. 13, 2013, which claimspriority to U.S. Provisional Application No. 61/844,804, filed on Jul.10, 2013, U.S. Provisional Application No. 61/840,403, filed on Jun. 27,2013, U.S. Provisional Application No. 61/800,223, filed on Mar. 15,2013, U.S. Provisional Application No. 61/762,435, filed on Feb. 8,2013, U.S. Provisional Application No. 61/737,374, filed on Dec. 14,2012, U.S. Provisional Application No. 61/683,192, filed on Aug. 14,2012; U.S. application Ser. No. 14/316,447, filed on Jun. 26, 2014, isalso a continuation-in-part of PCT International Patent Application No.PCT/US13/54797, filed on Aug. 13, 2013, which claims priority to U.S.Provisional Patent Application No. 61/683,192, filed on Aug. 14, 2012;U.S. Provisional Patent Application No. 61/737,374, filed on Dec. 14,2012; U.S. Provisional Patent Application No. 61/762,435, filed on Feb.8, 2013; U.S. Provisional Patent Application No. 61/800,223, filed onMar. 15, 2013; U.S. Provisional Patent Application No. 61/840,403 filedon Jun. 27, 2013; and U.S. Provisional Patent Application No. 61/844,804filed on Jul. 10, 2013, which applications are incorporated herein byreference in their entireties for all purposes. U.S. application Ser.No. 14/316,447, filed on Jun. 26, 2014, also claims the benefit of U.S.Provisional Patent Application No. 61/896,060 filed on Oct. 26, 2013;U.S. Provisional Patent Application No. 61/909,974 filed on Nov. 27,2013; U.S. Provisional Patent Application No. 61/937,344 filed on Feb.7, 2014; U.S. Provisional Patent Application No. 61/940,318 filed onFeb. 14, 2014; and U.S. Provisional Patent Application No. 61/991,018,filed on May 9, 2014, which applications are incorporated herein byreference in their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 30, 2018, isnamed 43487703322SL.txt and is 12 kilobytes in size.

BACKGROUND

Genomic sequencing can be used to obtain information in a wide varietyof biomedical contexts, including diagnostics, prognostics,biotechnology, and forensic biology. Sequencing may involve basicmethods including Maxam-Gilbert sequencing and chain-terminationmethods, or de novo sequencing methods including shotgun sequencing andbridge PCR, or next-generation methods including polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrentsemiconductor sequencing, HeliScope single molecule sequencing, SMRT®sequencing, and others. For most sequencing applications, a sample suchas a nucleic acid sample is processed prior to introduction to asequencing machine. A sample may be processed, for example, byamplification or by attaching a unique identifier. Often uniqueidentifiers are used to identify the origin of a particular sample.

SUMMARY

The present disclosure generally provides methods, compositions,devices, and kits for the generation of beads with covalently attachedpolynucleotides. Such beads may be used for any suitable application.The present disclosure further provides methods, compositions, devices,and kits for polynucleotide barcoding.

In an aspect, the present disclosure provides a method forpolynucleotide barcoding, comprising: (a) in a first set of partitions,attaching a first set of nucleic acid barcode molecules comprisingbarcode sequences to polynucleotides to provide a first set of barcodedpolynucleotides; (b) pooling contents of said first set of partitions;(c) partitioning at least a portion of said contents from (b) into asecond set of partitions different than said first set of partitions;and (d) in said second set of partitions, attaching a second set ofnucleic acid barcode molecules comprising barcode sequences to saidfirst set of barcoded polynucleotides to provide a second set ofbarcoded polynucleotides.

In some embodiments, a subset of barcoded polynucleotides of said firstset of barcoded polynucleotides have identical barcode sequences. Insome embodiments, at least a subset of barcoded polynucleotides of saidsecond set of barcoded polynucleotides have different barcode sequences.

In some embodiments, said polynucleotides are attached to beads.

In some embodiments, said polynucleotides are cellular polynucleotides.

In some embodiments, said first set of partitions are a first set ofwells and said second set of partitions are a second set of wells. Insome embodiments, said first set of partitions are a first set ofdroplets and said second set of partitions are a second set of droplets.

In some embodiments, said first set of nucleic acid barcode molecules orsaid second set of nucleic acid barcode molecules are attached to saidpolynucleotides by a nucleic acid reaction. In some embodiments, saidfirst set of nucleic acid barcode molecules or said second set ofnucleic acid barcode molecules are attached to said polynucleotides by anucleic acid primer extension reaction. In some embodiments, said firstset of nucleic acid barcode molecules or said second set of nucleic acidbarcode molecules are attached to said polynucleotides by a nucleic acidamplification reaction. In some embodiments, said first set of nucleicacid barcode molecules or said second set of nucleic acid barcodemolecules are attached to said polynucleotides by ligation.

In some embodiments, said first set of barcoded polynucleotides or saidsecond set of barcoded polynucleotides are coupled to a plurality ofbeads. In some embodiments, said nucleic acid barcode molecules arereleasable from said plurality of beads. In some embodiments, saidplurality of beads are a plurality of gel beads. In some embodiments,each gel bead of said plurality of gel beads is dissolvable ordisruptable.

In some embodiments, each nucleic acid barcode molecule of said firstset of nucleic acid barcode molecules or said second set of nucleic acidbarcode molecules further comprises a primer sequence. In someembodiments, said primer sequence is a targeted primer sequence or arandom primer sequence. In some embodiments, each nucleic acid barcodemolecule of said first set of nucleic acid barcode molecules or saidsecond set of nucleic acid barcode molecules further comprises a randomN-mer.

In some embodiments, said barcode sequences of said first set of nucleicacid barcode molecules or said barcode sequences of said second set ofnucleic acid barcode molecules are greater than 4 nucleic acid bases inlength. In some embodiments, said barcode sequences of said first set ofnucleic acid barcode molecules or said barcode sequences of said secondset of nucleic acid barcode molecules are 4 to 20 nucleic acid bases inlength. In some embodiments, each barcoded polynucleotide of said secondset of barcoded polynucleotides comprises a barcode sequence that is atleast 15 nucleic acid bases in length.

In some embodiments, said second set of barcoded polynucleotidescomprises at least 100,000 different barcode sequences. In someembodiments, said second set of barcoded polynucleotides comprises atleast 1,000,000 different barcode sequences. In some embodiments, saidsecond set of barcoded polynucleotides comprises at least 10,000,000different barcode sequences.

In some embodiments, the method further comprises: (e) pooling contentsof said second set of partitions; (f) partitioning at least a portion ofsaid contents from (e) into a third set of partitions different thansaid first set of partitions and said second set of partitions; and (g)in said third set of partitions, attaching a third set of nucleic acidbarcode molecules comprising barcode sequences to said second set ofbarcoded polynucleotides to provide a third set of barcodedpolynucleotides. In some embodiments, the method further comprises: (h)pooling contents of said second third of partitions; (i) partitioning atleast a portion of said contents from (h) into a fourth set ofpartitions different than said first set of partitions, said second setof partitions, and said third set of partitions; and (j) in said fourthset of partitions, attaching a fourth set of nucleic acid barcodemolecules comprising barcode sequences to said third set of barcodedpolynucleotides to provide a fourth set of barcoded polynucleotides.

In some embodiments, said first set of nucleic acid barcode moleculesare attached to said polynucleotides by a nucleic acid primer extensionreaction, wherein said second set of nucleic acid barcode molecules areattached to said first set of barcoded polynucleotides by ligation,wherein said third set of nucleic acid barcode molecules are attached tosaid second set of barcoded polynucleotides by ligation, and whereinsaid fourth set of nucleic acid barcode molecules are attached to saidthird set of barcoded polynucleotides by a nucleic acid amplificationreaction.

An aspect of the disclosure provides a method of barcoding samplematerials. A first partition comprising a plurality of nucleic acidbarcode molecules associated therewith may be provided and the nucleicacid barcode molecules can comprise the same nucleic acid barcodesequence. The first partition may be co-partitioned with components of asample material into a second partition and the barcode molecules canthen be released from the first partition into the second partition. Thereleased barcode molecules can be attached to one or more of thecomponents of the sample material or fragments thereof within the secondpartition. In some cases, the first partition may comprise at least1,000 barcode molecules, at least 10,000 barcode molecules, at least100,000 barcode molecules, or at least 1,000,000 barcode moleculesassociated therewith having the same barcode sequence. Moreover, in someexamples, the first partition may be a bead, a microcapsule, or adroplet. In some cases, the first partition may comprise a bead (e.g., agel bead) and the barcode molecules may be releasably coupled to thebead. Moreover, the second partition may comprise a droplet and/or maycomprise no more than one first partition.

In some cases, the co-partitioning of the first partition and thecomponents of the sample material into the second partition may comprisecombining a first aqueous fluid comprising beads with a second aqueousfluid comprising the sample components in a droplet within an immisciblefluid. Moreover, the barcode molecules may be released from the firstpartition by degrading the first partition. In cases where the firstpartition is a bead, the barcode molecules may be released in the secondpartition by degrading the bead and/or cleaving a chemical linkagebetween the barcode molecules and the bead. In some cases, at least oneof crosslinking of the bead and a linkage between the bead and thebarcode molecules may comprise a disulfide linkage. In such cases, thebarcode molecules may be released from the bead by exposing the bead toa reducing agent (e.g., dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP)).

The sample materials may comprise one or more template nucleic acidmolecules and the barcode molecules may be attached to one or morefragments of the template nucleic acid molecules. In some cases, thebarcode molecules may comprise a primer sequence complementary to atleast a portion of the template nucleic acid molecules and the barcodemolecules may be attached to the template nucleic acid molecule orfragments thereof by extending the barcode molecules to replicate atleast a portion of the template nucleic acid molecules. Moreover, thesample materials may comprise the contents of a single cell, such as,for example, a cancer cell or a bacterial cell (e.g., a bacterial cellisolated from a human microbiome sample).

Furthermore, a plurality of first partitions comprising a plurality ofdifferent nucleic acid barcode sequences may be provided. Each of thefirst partitions can include a plurality of at least 1000 nucleic acidbarcode molecules having the same nucleic acid barcode sequenceassociated therewith. The first partitions may be co-partitioned withcomponents of the sample material into a plurality of second partitions.The nucleic acid barcode molecules from the first partitions may then bereleased into the second partitions. The released nucleic acid barcodemolecules can then be attached to the components of the sample materialor fragments thereof within the second partitions. In some cases, theplurality of different nucleic acid barcode sequences may comprise atleast about 1,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 100,000 different barcodesequences, or at least about 500,000 different barcode sequences.Additionally, in some examples, a subset of the second partitions maycomprise the same nucleic acid barcode sequence. For example, at leastabout 1%, at least about 2%, or at least about 5% of the secondpartitions may comprise the same nucleic acid barcode sequence. Inaddition, in some cases, at least 50% of the second partitions, at least70% of the second partitions, or at least 90% of the second partitionsmay contain no more than one first partition. In some cases, at least50% of the second partitions, at least 70% of the second partitions, orat least 90% of the second partitions may contain exactly one firstpartition.

Fragments of the components of the sample material may include one ormore fragments of one or more template nucleic acid sequences. Thefragments of the template nucleic acid sequences may be sequenced andcharacterized based at least in part upon a nucleic acid barcodesequence attached thereto. In some cases, the fragments of the templatenucleic acid sequences may be characterized by mapping a fragment of anindividual template nucleic acid sequence of the template nucleic acidsequences to an individual template nucleic acid sequence of thetemplate nucleic acid sequences or a genome from which the individualtemplate nucleic acid sequence was derived. In some cases, the fragmentsof the template nucleic acid sequence may be characterized by at leastidentifying an individual nucleic acid barcode sequence of the differentnucleic acid barcode sequences and identifying a sequence of anindividual fragment of the fragments of the template nucleic acidsequences attached to the individual nucleic acid barcode sequence.

An additional aspect of the disclosure provides a method of barcodingsample materials. A plurality of first partitions may be provided thatcomprise a plurality of different nucleic acid barcode sequences. Eachof the first partitions may comprise a plurality of nucleic acid barcodemolecules having the same nucleic acid barcode sequence associatedtherewith. The first partitions may by co-partitioned with components ofa sample material into a plurality of second partitions. The barcodemolecules can be released from the first partitions into the secondpartitions. The released barcode molecules can then be attached to thecomponents of the sample material within the second partitions.

A further aspect of the disclosure provides a method of barcoding samplematerials. An activatable nucleic acid barcode sequence may be providedand partitioned with one or more components of a sample material into afirst partition. The activatable nucleic acid barcode sequence may beactivated to produce an active nucleic acid barcode sequence in thefirst partition. The active nucleic acid barcode sequence can beattached to the one or more components of the sample material. In somecases, the activatable nucleic acid barcode sequence may be activated byreleasing the activatable nucleic acid barcode sequence from a secondpartition within the first partition. In some cases, the activatablenucleic acid barcode sequence may be activated by removing a removableprotecting group from the activatable nucleic acid barcode sequence.

An additional aspect of the disclosure provides a composition comprisinga first partition that comprises one or more sample components and asecond partition that is contained within the first partition. Thesecond partition can have a plurality of oligonucleotides releasablyassociated therewith and the oligonucleotides may comprise a commonbarcode sequence. In some cases, the first partition may comprise anaqueous droplet in an emulsion and/or the second partition may comprisea microcapsule or bead. In some cases, the second partition may comprisea degradable bead that can be a photodegradable bead, a chemicallydegradable bead, and/or a thermally degradable bead. The degradable beadmay comprise a chemically cleavable cross-linking such as, for example,disulfide cross-linking. Moreover, in some cases, the oligonucleotidesmay be releasably associated with the second partition by a cleavablelinkage. The cleavable linkage may comprise, for example, a chemicallycleavable linkage, a photocleavable linkage, and/or a thermallycleavable linkage. In some cases, the cleavable linkage is a disulfidelinkage. Furthermore, the sample components may comprise, for example,nucleic acids (e.g., genomic nucleic acid such as genomic DNA) orfragments thereof. The nucleic acids can comprise nucleic acid fragmentsthat can have a length of between about 1 kb and about 100 kb, a lengthof between about 5 kb and about 50 kb, or a length of between about 10kb and about 30 kb.

In some cases, the composition comprises a plurality of first partitionsand a plurality of different second partitions. Each of the differentsecond partitions can be disposed within a separate first partition andmay comprise a plurality of oligonucleotides releasably associatedtherewith. The oligonucleotides associated with each second partitioncan comprise a common barcode sequence and the oligonucleotidesassociated with different second partitions can comprise differentbarcode sequences. In some cases, the different second partitions maycomprise at least 1,000 different second partitions, at least 10,000different second partitions, at least 100,000 different secondpartitions, or at least 500,000 different second partitions.

An additional aspect of the disclosure provides a method that comprisescombining a sample of nucleic acids with a library of barcoded beads toform a mixture. The mixture can be partitioned into a plurality ofpartitions such that at least a subset of the partitions comprises atmost one barcoded bead. Within the partitions, barcodes can be releasedfrom the barcoded beads. In some cases, the barcodes may bepre-synthesized with known sequences and/or may comprise a plurality ofrandom N-mers. The random N-mers may be hybridized to the sample ofnucleic acids in order to perform, for example, a nucleic acidamplification reaction within the partitions. In some cases, thebarcoded beads may be capable of being dissolved by a reducing agent andmay comprise disulfide bonds. Moreover, in some cases, the samplenucleic acids may be genomic DNA that may or may not be fragmented priorto being combined with the barcoded beads. In some cases, barcodes maybe released from the barcoded beads by the action of a reducing agent.In some cases, the barcoded beads may comprise a matrix that iscrosslinked with disulfide bonds and barcodes may be released from thebarcoded beads by the action of a reducing agent that dissolves thebarcoded beads. In some cases, barcodes may be released from thebarcoded beads by heating the partitions.

In some cases, the sample of nucleic acids may be combined with thelibrary of barcoded beads and/or the mixture of the two may bepartitioned into a plurality of partitions using a microfluidic device.In some examples, the partitions may be aqueous droplets within awater-in-oil emulsion. Partitioning of the mixture into aqueous dropletswithin a water-in-oil emulsion may be completed using a microfluidicdevice.

A microfluidic device may be a droplet generator and, in some cases, maycomprise a first input channel and a second input channel that meet at ajunction that is fluidly connected to an output channel. The sample ofnucleic acids can be introduced into the first input channel and thelibrary of barcoded beads can be introduced to the second input channelto generate the mixture of the sample nucleic acids and the library ofbarcoded beads in the output channel. In some cases, a reducing agentmay also be introduced to either or both of the first input channel andsecond input channel. Moreover, the first input channel and the secondinput channel may form a substantially perpendicular angle between oneanother.

In some cases, the output channel may be fluidly connected to a thirdinput channel at a junction. Oil can be introduced into the third inputchannel such that aqueous droplets within a water-in-oil emulsion andthat comprise barcoded beads are formed. The droplets may comprise onaverage, for example, at most ten barcoded beads, at most seven barcodedbeads, at most five barcoded beads, at most three barcoded beads, atmost two barcoded beads, or at most one barcoded bead. Moreover, themicrofluidic device may comprise a fourth input channel that intersectsthe third input channel and the output channel at a junction. In somecases, oil may also be provided to the fourth input channel. In somecases, the microfluidic device may include an additional input channelthat intersects the first input channel, the second input channel, orthe junction of the first input channel and the second input channel. Insome cases, a reducing agent may be introduced into the additional inputchannel.

An additional aspect of the disclosure provides a composition comprisinga bead that is covalently linked to a plurality of oligonucleotides thatcomprise an identical barcode sequence and a variable domain. In somecases, the oligonucleotides may also comprise a primer binding siteand/or a universal primer. Additionally, the identical barcode sequencemay be between about 6 nucleotides and about 20 nucleotides in length.Moreover, the oligonucleotides may be covalently linked to the bead bydisulfide linkages and/or the bead may comprise a cystamine or amodified cystamine. In some cases, the bead may be capable of beingsubstantially dissolved by a reducing agent. Furthermore, in some cases,the bead may comprise at least about 1,000,000 oligonucleotidescomprising an identical barcode sequence. In some cases, at least about30% of the oligonucleotides may comprise variable domains with differentsequences. In some cases, the variable domain may be a random N-mer. Insome cases, the bead may be covalently linked to the oligonucleotidesthrough a cleavable linkage such as, for example, a chemically cleavablelinkage, a photocleavable linkage, and a thermally cleavable linkage.

A further aspect of the disclosure provides a composition comprising abead that may comprise a plurality of more than 1,000,000oligonucleotides, where each of the oligonucleotides comprises aconstant region and a variable region. The bead can be capable of beingsubstantially dissolved with a reducing agent. In some cases, each ofthe oligonucleotides may comprise an identical constant region. In somecases, at least 25% of the oligonucleotides may have an identicalconstant region. In some cases, the constant region may be a barcodesequence. In some cases, at least 25% of the oligonucleotides may have avariable region comprising a different sequence. A further aspect of thedisclosure provides a library comprising at least about 1,000,000 beadsthat each comprise a plurality of more than 1,000,000 oligonucleotidesthat comprise a constant region and a variable region. In some cases, atleast about 25% of the beads comprise oligonucleotides with differentnucleotide sequences.

An additional aspect of the disclosure provides a composition comprisinga plurality of beads where each of the beads comprises a plurality ofoligonucleotides releasably coupled thereto. The oligonucleotidesassociated with an individual bead may comprise a common barcode domainand a variable domain. The common barcode domain can be differentbetween two or more of the beads. In some cases, the beads may compriseat least about 10,000 different barcode domains coupled to differentbeads. In some cases, each of the beads may comprise at least about1,000,000 oligonucleotides releasably coupled thereto.

A further aspect of the disclosure provides a method of generatingfunctionalized beads. A plurality of polymers or monomers may be mixedwith one or more oligonucleotides. The polymers or monomers can becrosslinked such that disulfide bonds form between the polymers ormonomers, thereby forming hardened beads. Moreover, covalent linkagescan be caused to form between the oligonucleotides and the polymers ormonomers. In some cases, the polymers or monomers may compriseacrylamide. In some cases, the polymers and monomers may be crosslinkedto form hardened beads and covalent linkages can be caused to formbetween the oligonucleotides and the polymers or monomers eithercontemporaneously or sequentially. Moreover, in some cases, theoligonucleotides may comprise a primer (e.g., a universal primer, asequencing primer) that may be linked to an acrydite moiety.

Additionally, one or more additional oligonucleotides may be attached tothe oligonucleotides. The additional oligonucleotides may be a barcodesequence and, thus, upon attachment to the oligonucleotides, barcodedbeads can be formed. In some cases, the barcode sequence may be betweenabout 6 nucleotides and about 20 nucleotides in length.

In some cases, functionalized beads may be combined with a plurality offirst additional oligonucleotides to create a mixture. The mixture maybe partitioned into a plurality of partitions such that, on average,each partition comprises no more than one of the first additionaloligonucleotides. In some cases, the partitions may be aqueous dropletswithin a water-in-oil emulsion and/or may be generated by a microfluidicdevice. In some cases, the partitions are generated by a bulkemulsification process. Moreover, the first additional oligonucleotidescan be amplified within the partitions to produce beads comprisingamplified first oligonucleotides. In some cases, a capture primer may beused during amplification and the capture primer may be attached to acapture moiety such as, for example, biotin, streptavidin orglutathione-S-transferase (GST). Following amplification, the contentsof the partitions can be pooled into a common vessel. The beadscomprising amplified first oligonucleotides can be separated from thecontents of the partitions. In some cases, a probe may be hybridized tothe amplified first oligonucleotides. The probe may comprise a capturemoiety.

Furthermore, one or more second additional oligonucleotides can beattached to the amplified first oligonucleotides. In some cases, thesecond additional oligonucleotides may comprise a random N-mer sequenceand/or a pseudo random N-mer sequence. In some cases, the secondadditional oligonucleotides may comprise a primer binding site that cancomprise a universal sequence portion. In some cases, the primer bindingsite may comprise uracil containing nucleotide. Moreover, the universalsequence portion can be compatible with a sequencing device and/or maycomprise a subsection of uracil containing nucleotides.

An additional aspect of the disclosure provides a method of preparing abarcode library. A plurality of separate first bead populations can beprovided and a first oligonucleotide comprising a first barcode sequencesegment can be attached to the separate first bead populations, suchthat each separate first bead population comprises a different firstbarcode sequence segment attached thereto. The separate bead populationscan then be pooled to provide a first pooled bead population. The firstpooled bead population can then be separated into a plurality of secondbead populations. A second oligonucleotide comprising a second barcodesequence segment may be attached to the first oligonucleotide attachedto the second bead populations, such that each of the separate secondbead populations comprises a different second barcode sequence segment.The separate second bead populations can then be pooled to provide asecond pooled bead population that comprises a barcode library.

In some cases, the first barcode sequence segments and the secondbarcode sequence segments may be independently selected from a first setof barcode sequence segments. Additionally, the first barcode sequencesegments and the second barcode sequence segments may independentlycomprise at least 4 nucleotides in length, at least 6 nucleotides inlength, or at least 10 nucleotides in length. In some cases, the firstbarcode sequence segments and the second barcode sequence segments mayindependently include from about 4 nucleotides in length to about 20nucleotides in length. Moreover, in some cases, the first beadpopulations may comprise at least 100 different first barcode sequencesegments or at least 1,000 different first barcode sequence segments.Furthermore, in some cases, at least 1,000,000 first oligonucleotidemolecules may be attached to each bead in each of the separate firstbead populations. In some cases, the second bead populations maycomprise at least 100 different second barcode sequence segments or atleast 1,000 different second barcode sequence segments. In some cases,at least 1,000,000 second oligonucleotide molecules may be attached toeach bead in each of the second bead populations.

Further, in some cases, at least one of the first oligonucleotide andthe second oligonucleotide may comprise a functional sequence such as,for example, a primer sequence, a primer annealing sequence, anattachment sequence, and a sequencing primer sequence. In some cases, atleast one of the first oligonucleotide and the second oligonucleotidemay comprise a sequence segment that comprises one or more of a uracilcontaining nucleotide and a non-native nucleotide.

In some cases, the first oligonucleotide may be attached to the separatefirst bead populations by providing a splint sequence that is in partcomplementary to at least a portion of the first oligonucleotide and inpart complementary to at least a portion of an oligonucleotide attachedto the separate first bead populations. In some cases, the firstoligonucleotide may be attached to the separate first bead populationssuch that it is releasably attached to the separate first beadpopulations. For example, the first oligonucleotide may be attached tothe separate first bead populations through a cleavable linkage. In somecases, the first oligonucleotide may be attached to the separate firstbead populations either directly or indirectly.

Additionally, in some cases, the second oligonucleotide may be attachedto the first oligonucleotide by ligation. In some cases, the secondoligonucleotide may be attached to the first oligonucleotide byproviding a splint sequence that is in part complementary to at least aportion of the first oligonucleotide and in part complementary to atleast a portion of the second oligonucleotide. In some cases, the splintsequence may provides a first overhang sequence when hybridized to thefirst oligonucleotide, and the second barcode sequence segment maycomprise a second overhang sequence complementary to the first overhangsequence. In some cases, the first overhang sequence and the secondoverhang sequences may be from about 2 nucleotides in length to about 6nucleotides in length. Furthermore, in some cases, the first overhangsequence may comprise a plurality of different overhang sequences, andthe second oligonucleotides may comprise a plurality of different secondoverhang sequences complementary to the plurality of different firstoverhang sequences.

Moreover, the separate first bead populations may comprise degradablebeads, such as, for example, chemically degradable beads,photodegradable beads, and/or thermally degradable beads. In some cases,the separate first bead populations may comprise beads that comprisechemically reducible cross-linkers such, as for example, chemicallyreducible cross-linkers that comprise disulfide linkages.

In some cases, a third oligonucleotide may be attached to the secondoligonucleotide attached to the first oligonucleotide. The thirdoligonucleotide may comprise a functional sequence that may be a primersequence (e.g., a universal primer sequence, a targeted primer sequence,or a random sequence) and/or may be a random N-mer sequence. In caseswhere the third oligonucleotide comprises a random N-mer sequence, therandom N-mer sequence may be from about 5 nucleotides in length to about25 nucleotides in length.

An additional aspect of the disclosure provides a method of preparing abarcode library. A first pooled bead population comprising a pluralityof different first bead populations may be provided, where eachdifferent first bead population comprises a different firstoligonucleotide attached thereto. Each different first oligonucleotidemay comprise a different first barcode sequence segment. The firstpooled bead population may be separated into a plurality of second beadpopulations. A second oligonucleotide comprising a second barcodesequence segment may be attached to the first oligonucleotide alreadyattached to the second bead populations, where each second beadpopulation comprises a different second barcode sequence segment. Thesecond bead populations can be pooled to provide a second pooled beadpopulation comprising a barcode library.

In some cases, the first oligonucleotide may be releasably attached tothe beads in the first pooled bead population. In some cases, the firstoligonucleotide may be attached to the beads in the first pooled beadpopulation through a cleavable linkage. In some cases, the beads in thefirst pooled population may each comprise at least 1,000,000 firstoligonucleotides attached thereto. In some cases, the first pooled beadpopulation may comprise at least 10 different first bead populations, atleast 100 different first bead populations, or at least 500 differentfirst bead populations.

A further aspect of the disclosure provides a barcode library comprisinga plurality of different oligonucleotides. Each differentoligonucleotide may comprise a first barcode sequence segment selectedfrom a first set of barcode sequence segments; a second barcode sequencesegment selected from a second set of barcode sequence segments; and alinking sequence joining the first barcode sequence segment and thesecond barcode sequence segment. The linking sequence can be from about2 nucleotides in length to about 6 nucleotides in length and may beselected from a set of linking sequences. In some cases, the set oflinking sequences includes from about 2 different linking sequences toabout 50 different linking sequences. In some cases, the first set ofbarcode sequence segments and the second set of barcode sequencesegments are the same.

An additional aspect of the disclosure provides a method of amplifying atemplate nucleic acid sequence. A template nucleic acid sequence and abead comprising a plurality of releasably attached oligonucleotides maybe co-partitioned into a partition. The oligonucleotides may comprise aprimer sequence complementary to one or more regions of the templatenucleic acid sequence and may comprise a common sequence. The primersequence can be annealed to the template nucleic acid sequence and theprimer sequence can be extended to produce one or more first copies ofat least a portion of the template nucleic acid sequence, where the oneor more first copies comprising the primer sequence and the commonsequence.

In some cases, the primer sequence may comprise a variable primersequence (e.g., a random N-mer) and/or may comprise a targeted primersequence. In some cases, the partition may comprise a droplet in anemulsion. Prior to annealing the primer sequence to the template nucleicacid sequence, the oligonucleotides may be released from the bead intothe partition. In some examples, a polymerase enzyme (e.g., anexonuclease deficient polymerase enzyme) may be provided in thepartition. Moreover, extension of the primer sequence may compriseextending the primer sequence using a strand displacing polymeraseenzyme (e.g., a thermostable strand displacing polymerase enzyme having,for example, substantially no exonuclease activity). Furthermore, theoligonucleotides may be exonuclease resistant. For example, theoligonucleotides may comprise one or more phosphorothioate linkages. Insome cases, the phosphorothioate linkages may comprise aphosphorothioate linkage at a terminal internucleotide linkage in theoligonucleotides.

Additionally, one or more variable primer sequences may be annealed tothe first copies and extended to produce one or more second copies fromthe first copies, such that the second copies comprise the one or morevariable primer sequences and the common sequence. In some cases, thesecond copies may comprise a sequence complementary to at least aportion of an individual first copy of the first copies and a sequencecomplementary to an individual variable sequence of the one or morevariable primer sequences. In some cases, the second copies maypreferentially form a hairpin molecule under annealing conditions.Moreover, in some cases, the oligonucleotides may comprise a sequencesegment that is not copied during the extension of the variable primersequences. The sequence segment that is not copied may comprise, forexample, one or more uracil containing nucleotides. In addition, anysteps of the method may be repeated to produce amplified nucleic acids.

A further aspect of the disclosure provides a method of amplifying aplurality of different nucleic acids. Different nucleic acids may bepartitioned into separate first partitions, where each first partitioncomprises a second partition having a plurality of oligonucleotidesreleasably associated therewith. The plurality of oligonucleotidesassociated with a given second partition may comprise a variable primersequence and a barcode sequence, with the oligonucleotides associatedwith different second partitions comprising different barcode sequences.The oligonucleotides associated with the plurality of second partitionscan be released into the first partitions. The variable primer sequencesin the first partitions can be released to nucleic acids within thefirst partitions and extended to produce one or more copies of at leasta portion of the nucleic acids within the first partitions, such thatthe copies comprise the oligonucleotides and associated barcodesequences released into the first partitions. In some cases, the firstpartitions may comprise droplets in an emulsion and the secondpartitions may comprise beads. In some cases, each bead may comprisemore than 100,000 oligonucleotides associated therewith or more than1,000,000 oligonucleotides associated therewith. In some cases, thesecond partitions may comprise at least 1,000 different barcodesequences, at least 10,000 different barcode sequences, or at least100,000 different barcode sequences.

An additional aspect of the disclosure provides a method of whole genomeamplification. A random primer may be hybridized to a genomic nucleicacid. The random primer may be attached to a universal nucleic acidsequence and a nucleic acid barcode sequence, where the universalnucleic acid sequence may comprise one or more uracil containingnucleotides. The random primer may be extended to form an amplifiedproduct and the amplified product may be exposed to conditions suitableto cause the amplified product to undergo an intramolecularhybridization reaction that forms a partial hairpin molecule. In somecases, the random primer may be a random N-mer sequence. In some cases,the universal nucleic acid sequence may comprise a segment of at least10 nucleotides that do not comprise uracil. Moreover, the method may beperformed in the presence of an oligonucleotide blocker. Theoligonucleotide blocker may be capable of hybridizing to at least aportion of the universal nucleic acid sequence and/or may comprise a C3spacer (/3SpC3/), a Dideoxy-C(/3ddC/), or a 3′ phosphate.

An additional aspect of the disclosure provides a method of amplifyingnucleic acids. A genomic component may be fragmented into a plurality offirst fragments. The first fragments may be co-partitioned with aplurality of oligonucleotides into a plurality of partitions. Theoligonucleotides in each of the partitions may comprise a primersequence and a common sequence. The primer sequences in each partitionmay be annealed to a plurality of different regions of the firstfragments within each partition and the primer sequences extended alongthe first fragments to produce amplified first fragments within eachpartition. In some cases, the amplified first fragments within thepartitions may comprise at least 1× coverage of the genomic component,at least 2× coverage of the genomic component, or at least 10× coverageof the genomic component. In some cases, the genomic component maycomprise a chromosome. In some cases, the genomic component may comprisea whole genome of an organism.

A further aspect of the disclosure provides a method of characterizing anucleic acid segment. A nucleic acid segment may be co-partitioned witha bead comprising a comprising a plurality of oligonucleotides thatcomprise a common nucleic acid barcode sequence into a partition. Theoligonucleotides may be attached to fragments of the nucleic acidsegment or to copies of portions of the nucleic acid segment, such thatthe common nucleic acid barcode sequence is attached to the fragments ofthe nucleic acid segment or the copies of the portions of the nucleicacid segment. The fragments of the nucleic acid segment or the copies ofthe portions of the nucleic acid segment and attached common nucleicacid barcode sequence can be sequenced and the fragments of the nucleicacid segment or the copies of the nucleic acid segment can becharacterized as being linked within the nucleic acid segment based atleast in part, upon a their attachment to the common nucleic acidbarcode sequence. The nucleic acid segment and the bead, for example,may be co-partitioned into a droplet in an emulsion or may beco-partitioned into a microcapsule. In some cases, the fragments of thenucleic acid segment may comprise overlapping fragments of the nucleicacid segment. In some cases, the fragments of the nucleic acid segmentmay comprise greater than 2× coverage of the nucleic acid segment orgreater than 10× coverage of the nucleic acid segment.

Moreover, in some cases, the oligonucleotides may be releasably attachedto the bead. For example, the oligonucleotides may be releasable fromthe bead upon the application of a stimulus (e.g., a thermal stimulus, aphoto stimulus, a chemical stimulus, etc.) to the bead. In some cases,the application of the stimulus may result in the cleavage of a linkagebetween the oligonucleotides and the bead and/or may result in thedegradation of the bead, such that the oligonucleotides are releasedfrom the bead. Furthermore, the bead may comprise at least about 10,000oligonucleotides attached thereto, at least about 100,000oligonucleotides attached thereto, at least about 1,000,000oligonucleotides attached thereto, at least about 10,000,000oligonucleotides attached thereto, or at least about 100,000,000oligonucleotides attached thereto. Additionally, in some cases, theoligonucleotides may comprise one or more functional sequences, such as,for example, a primer sequence, a primer annealing sequence, or animmobilization sequence. In some cases, the fragments of the nucleicacid segment or the copies of the portions of the nucleic acid segmentand attached common nucleic acid barcode sequence may be sequenced via asequencing by synthesis process.

Further, in some cases, the oligonucleotides may comprise a primersequence capable of annealing with a portion of the nucleic acid segmentor a complement thereof. The primer sequence can be extended toreplicate at least a portion of the nucleic acid segment or complementthereof, to produce a copy of a portion of the nucleic acid segment orcomplement thereof that comprises the common nucleic acid barcodesequence. In some cases, the oligonucleotides may comprise at least afirst sequencing primer sequence.

In some cases, a plurality of nucleic acid segments may beco-partitioned with a plurality of different beads into a plurality ofseparate partitions, such that each partition of a plurality ofdifferent partitions of the separate partitions contains a single bead.Each bead may comprise a plurality of oligonucleotides that comprise acommon barcode sequence attached thereto, where the different beadscomprises a plurality of different barcode sequences. Barcode sequencesin each partition may be attached to fragments of the nucleic acidsegments or to copies of portions of the nucleic acid segments withinthe separate partitions. The fragments or copies can then be pooled fromthe separate partitions and the fragments or copies and any associatedbarcode sequences may be sequenced to provide sequenced fragments orsequenced copies. The sequenced fragments or sequenced copies may becharacterized as deriving from a common nucleic acid segment, based inpart upon the sequenced fragments or sequenced copies comprising acommon barcode sequence. In some cases, the nucleic acid segments maycomprise fragments of at least a portion of a genome. In such cases,sequences may be assembled from the sequenced fragments or sequencedcopies to provide a contiguous sequence of the at least a portion of thegenome. Assembly of the sequences from the sequenced fragments orsequenced copies may be based in part upon each of a nucleotide sequenceof the sequenced fragmentsor sequenced copies and the sequencedfragments or sequenced copies comprising a common barcode sequence.Moreover, in some cases, the fragments of the nucleic acid segments orthe copies of the portions of the nucleic acid segments may becharacterized based in part upon each of a nucleotide sequence of thefragments of the nucleic acid segments or the copies of the portions ofthe nucleic acid segments and the sequenced fragments or sequencedcopies comprising a common barcode sequence.

In some cases, the different beads may comprise at least 1,000 differentbarcode sequences, at least 10,000 different barcode sequences, at least100,000 different barcode sequences, or at least 1,000,000 differentbarcode sequences. In some cases, two or more partitions of the separatepartitions may comprise beads that comprise the same barcode sequence.In some cases, at least 1% of the separate partitions comprise beadshaving the same barcode sequence.

An additional aspect of the disclosure provides a method ofcharacterizing a target nucleic acid. First fragments of a targetnucleic acid may be partitioned into a plurality of droplets, where eachdroplet comprises a bead having a plurality of oligonucleotides attachedthereto. The oligonucleotides attached to a given bead can comprise acommon barcode sequence. The common barcode sequence can be attached tosecond fragments of the first fragments and the droplets can be pooled.The second fragments and attached barcode sequences can sequenced andthe second fragments can be mapped to one or more of the first fragmentsbased, at least in part, upon the second fragments comprising a commonbarcode sequence.

An additional aspect of the disclosure provides a method of sequencingnucleic acids. A plurality of target nucleic acid sequences may beprovided and separated into a plurality of separate partitions. Eachpartition of the separate partitions may comprise one or more targetnucleic acid sequences and a bead comprising a plurality ofoligonucleotides attached thereto. The oligonucleotides attached to agiven bead may comprise a common barcode sequence. The oligonucleotidesmay be attached to fragments of the one or more target nucleic acidsequences or to copies of portions of the one or more target nucleicacid sequences within a partition, thereby attaching the common barcodesequence to the fragments of the one or more target nucleic acidsequences or the copies of the portions of the one or more targetnucleic acid sequences. The separate partitions can be pooled and thefragments of the one or more target nucleic acid sequences or the copiesof the portions of the one or more target nucleic acid sequences andattached barcode sequences can be sequenced to provide barcoded fragmentsequences or barcoded copy sequences. In some cases, the barcodedfragment sequences or barcoded copy sequences can be assembled into oneor more contiguous nucleic acid sequences based, in part, upon a barcodeportion of the barcoded fragment sequences or barcoded copy sequences.

An additional aspect of the disclosure provides a method ofcharacterizing a nucleic acid segment. A nucleic acid segment may beco-partitioned with a bead comprising a plurality of oligonucleotidesthat comprise a common nucleic acid barcode sequence, into a firstdroplet. The oligonucleotides may be attached to fragments of thenucleic acid segment or to copies of portions of the nucleic acidsegment, thereby attaching the common nucleic acid barcode sequence tothe fragments of the nucleic acid segment or to the copies of theportions of the nucleic acid segment. The fragments of the nucleic acidsegment or the copies of the portions of the nucleic acid segment andattached common nucleic acid barcode sequence can be sequenced toprovide a plurality of barcoded fragment sequences or barcoded copysequences. The barcoded fragment sequences or barcoded copy sequencescan be assembled into one or more contiguous nucleic acid sequencesbased at least in part on the common nucleic acid barcode sequence. Insome cases, the barcoded fragment sequences or barcoded copy sequencesmay be assembled based in part upon a nucleic acid sequence ofnon-barcode potion of the barcoded fragment sequences or barcoded copysequences.

An additional aspect of the disclosure provides a method of sequencingnucleic acids. A plurality of target nucleic acid sequences may beprovided and the target nucleic acid sequences separated into aplurality of separate partitions. Each partition of the separatepartitions may comprise one or more target nucleic acid sequences and aplurality of oligonucleotides. The oligonucleotides in a given partitionmay comprise a common barcode sequence and the plurality of separatepartitions may comprise at least 10,000 different barcode sequences. Thecommon barcode sequence in each partition may be attached to fragmentsof the one or more target nucleic acid sequences or to copies ofportions of the one or more target nucleic acid sequences within thepartition. The separate partitions can be pooled and the fragments ofthe one or more target nucleic acid sequences or the copies of theportions of the one or more target nucleic acid sequences and attachedbarcode sequences can be sequenced. In some cases, the separatepartitions may comprise at least 100,000 different barcode sequences.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesfor all purposes and to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a flow diagram for making barcoded beads.

FIG. 1B is a flow diagram for processing a sample for sequencing.

FIG. 2 is a flow diagram for making beads.

FIG. 3A is a flow diagram for adding barcodes to beads by limitingdilution.

FIG. 3B is a flow diagram for adding additional sequences tooligonucleotides attached to beads.

FIGS. 4A-4N are diagrams for attaching sequences to beads. “g/w” meansgel-in-water; “g/w/o” means gel-in-water-in-oil;

FIGS. 5A-5E provides an illustration of a gel bead attached to anoligonucleotide (FIG. 5A), an image of a microfluidic chip used to makeGel Beads in Emulsions (GEM) (FIG. 5B), as well as images of GEMs (FIGS.5C, 5D, and 5E).

FIGS. 6A-6F provides bright-field (FIGS. 6A, 6C, and 6E) and fluorescent(FIGS. 6B, 6D, and 6F) images of beads with attached oligonucleotides.

FIGS. 7A-7C provide fluorescent images of beads attached to DNA.

FIGS. 8A-F provide images of barcode-enriched populations of beads.

FIGS. 9A-9D provide images of the dissolution of beads by heating.

FIG. 10A provides a schematic of a functionalized bead. FIGS. 10B-10Gprovide images of beads dissolved with a reducing agent.

FIG. 11A provides a schematic of a functionalized bead. FIGS. 11B-11Dprovide graphic depictions of the presence of barcode oligonucleotidesand primer-dimer pairs when beads are prepared using differentconditions.

FIG. 12 is a graphic depiction of content attached to beads.

FIG. 13A is a flow diagram illustrating the addition of barcodes tobeads using partitions.

FIG. 13B is a flow diagram illustrating the addition of additionalsequences to beads.

FIG. 13C is a diagram illustrating the use of a combinatorial approachin microwell plates to make barcoded beads.

FIGS. 14A-14C are diagrams of oligonucleotides containing universalsequences (R1, P5) and uracil containing nucleotides.

FIGS. 15A-15G are diagrams of steps used in the partial hairpinamplification for sequencing (PHASE) process.

FIG. 16A is a graphic depiction of including uracil containingnucleotides in the universal portion of the primer.

FIG. 16B is a graphic depiction of controlling amplification productlength by including acyNTPs in the reaction mixture.

FIG. 17 is a graphic depiction of reducing start site bias by adding ablocker oligonucleotide.

FIG. 18 is a flow diagram of a digital processor and its relatedcomponents.

FIG. 19 is a table providing example sequences for Illumina sequencers.FIG. 19 discloses SEQ ID NOS 4 and 7-9, respectively, in order ofappearance.

FIG. 20 is a table providing a list of example capture moietyconcentrations used to label beads.

FIG. 21 is a table providing a list of sequencing metrics obtained usingprimers comprising thymine containing nucleotides.

FIG. 22 is a table providing a list of sequencing metrics obtained usingprimers comprising uracil containing nucleotides.

FIGS. 23A-23D are schematics illustrating the use of an exampleligation-based combinatorial approach to make barcoded beads. FIGS.23A-23D disclose SEQ ID NOS 4, 10, 11, 12, 11, 13, 11 and 13,respectively, in order of appearance.

FIGS. 24A-24B are schematics illustrating an example use of spacer basesin a ligation-based combinatorial approach to make barcoded beads. FIGS.24A-24B disclose SEQ ID NOS 14, 14, 14 and 14-16, respectively, in orderof appearance.

FIGS. 25A-25C are schematics illustrating the use of an exampleligation-based combinatorial approach to make barcoded beads. FIGS.25A-25C disclose SEQ ID NOS 10, 17, 12, 17, 18 and 17, respectively, inorder of appearance.

FIG. 26 is a schematic illustrating example nucleic acids used in anexample ligation-based combinatorial approach to make barcoded beads.FIG. 26 discloses SEQ ID NOS 10, 19, 10, 20, 10, 21, 10 and 22,respectively, in order of appearance.

FIG. 27 is a schematic illustrating an example ligation-basedcombinatorial approach to make barcoded beads.

FIGS. 28A-28B are schematic representations of example targeted barcodeconstructs suitable for strand-specific amplification.

FIGS. 29A-29C are structural depictions of example monomers andcross-linkers that can be polymerized to generate beads.

FIGS. 30A-30C are structural depictions of an example method that can beused to generate beads.

FIG. 31 is a schematic depiction of example beads comprising functionalgroups that can be used to attach species to the beads.

FIG. 32 provides structural depictions of example initiators that may beused during a polymerization reaction.

FIG. 33A is a schematic depiction of barcode primers (SEQ ID NOS 23 and24, respectively, in order of appearance). FIGS. 33B-33E are graphicdepictions of data corresponding to example amplification reactionexperiments described in Example 16.

FIGS. 34A-34C are schematics of example hairpin constructs.

FIGS. 35A-35B are schematics of example methods for functionalizingbeads.

FIG. 36 is a photograph of a gel obtained during a gel electrophoresisexperiment described in Example 17.

FIG. 37A is a schematic depiction of oligonucleotides described inExample 18. FIG. 37B is a photograph of a gel obtained during a gelelectrophoresis experiment described in Example 18. FIG. 37C is amicrograph of beads obtained during a fluorescence microscopy experimentdescribed in Example 18.

FIGS. 38A-38F provide a schematic illustration of an exemplary nucleicacid barcoding and amplification process.

FIG. 39 provides a schematic illustration of an exemplary application ofthe methods described herein to nucleic acid sequencing and assembly.

FIG. 40 presents examples of alternative processing steps followingbarcoding and amplification of nucleic acids, as described herein.

DETAILED DESCRIPTION I. General Overview

This disclosure provides methods, systems and compositions useful in theprocessing of sample materials through the controlled delivery ofreagents to subsets of sample components, followed by analysis of thosesample components employing, in part, the delivered reagents. In manycases, the methods and compositions are employed for sample processing,particularly for nucleic acid analysis applications, generally, andnucleic acid sequencing applications, in particular. Included withinthis disclosure are bead compositions that include diverse sets ofreagents, such as diverse libraries of beads attached to large numbersof oligonucleotides containing barcode sequences, and methods of makingand using the same.

Methods of making beads can generally include, e.g. combining beadprecursors (such as monomers or polymers), primers, and cross-linkers inan aqueous solution, combining said aqueous solution with an oil phase,sometimes using a microfluidic device or droplet generator, and causingwater-in-oil droplets to form. In some cases, a catalyst, such as anaccelerator and/or an initiator, may be added before or after dropletformation. In some cases, initiation may be achieved by the addition ofenergy, such, as for example via the addition of heat or light (e.g., UVlight). A polymerization reaction in the droplet can occur to generate abead, in some cases covalently linked to one or more copies of anoligonucleotide (e.g., primer). Additional sequences can be attached tothe functionalized beads using a variety of methods. In some cases, thefunctionalized beads are combined with a template oligonucleotide (e.g.,containing a barcode) and partitioned such that on average one or fewertemplate oligonucleotides occupy the same partition as a functionalizedbead. While the partitions may be any of a variety of different types ofpartitions, e.g., wells, microwells, tubes, vials, microcapsules, etc.,in preferred aspects, the partitions may be droplets (e.g., aqueousdroplets) within an emulsion. The oligonucleotide (e.g., barcode)sequences can be attached to the beads within the partition by areaction such as a primer extension reaction, ligation reaction, orother methods. For example, in some cases, beads functionalized withprimers are combined with template barcode oligonucleotides thatcomprise a binding site for the primer, enabling the primer to beextended on the bead. After multiple rounds of amplification, copies ofthe single barcode sequence are attached to the multiple primersattached to the bead. After attachment of the barcode sequences to thebeads, the emulsion can be broken and the barcoded beads (or beadslinked to another type of amplified product) can be separated from beadswithout amplified barcodes. Additional sequences, such as a randomsequence (e.g., a random N-mer) or a targeted sequence, can then beadded to the bead-bound barcode sequences, using, for example, primerextension methods or other amplification reactions. This process cangenerate a large and diverse library of barcoded beads.

FIG. 1A illustrates an example method for generating a barcoded bead.First, gel precursors (e.g., linear polymers and/or monomers),cross-linkers, and primers may be combined in an aqueous solution, 101.Next, in a microfluidic device, the aqueous solution can then becombined with an oil phase, 102. Combining the oil phase and aqueoussolution can cause water-in-oil droplets to form, 103. Withinwater-in-oil droplets, polymerization of the gel precursors occurs toform beads comprising multiple copies of a primer, 104. Followinggeneration of a primer-containing bead, the emulsion may be broken, 105and the beads recovered. The recovered beads may be separated fromunreacted components, via, for example, washing and introduced to anysuitable solvent (e.g., an aqueous solvent, a non-aqueous solvent). Insome cases, the primer-containing beads may then be combined (e.g., vialimiting dilution methods) with template barcode sequences in dropletsof another emulsion, such that each droplet comprises on average atleast one bead and on average one or less molecules of a templatebarcode sequence. The template barcode sequence may be clonallyamplified, using the primer attached to the bead, resulting inattachment to the bead of multiple copies of a barcode sequencecomplementary to the template, 106. The barcoded beads may then bepooled into a population of beads either containing barcodes or notcontaining barcodes, 107. The barcoded beads may then be isolated by,for example, an enrichment step. The barcode molecules may also beprovided with additional functional sequence components for exploitationin subsequent processing. For example, primer sequences may beincorporated into the same oligonucleotides that include the barcodesequence segments, to enable the use of the barcode containingoligonucleotides to function as extension primers for duplicating samplenucleic acids, or as priming sites for subsequent sequencing oramplification reactions. In one example, random N-mer sequences may thenbe added to the barcoded beads, 108, via primer extension or otheramplification reaction and a diverse library of barcoded beads, 110, maythereby be obtained, where such random n-mer sequences can provide auniversal primer sequence. Likewise, functional sequences may includeimmobilization sequences for immobilizing barcode containing sequencesonto surfaces, e.g., for sequencing applications. For ease ofdiscussion, a number of specific functional sequences are describedbelow, such as P5, P7, R1, R2, sample indexes, random Nmers, etc., andpartial sequences for these, as well as complements of any of theforegoing. However, it will be appreciated that these descriptions arefor purposes of discussion, and any of the various functional sequencesincluded within the barcode containing oligonucleotides may besubstituted for these specific sequences, including without limitation,different attachment sequences, different sequencing primer regions,different n-mer regions (targeted and random), as well as sequenceshaving different functions, e.g., secondary structure forming, e.g.,hairpins or other structures, probe sequences, e.g., to allowinterrogation of the presence or absence of the oligonucleotides or toallow pull down of resulting amplicons, or any of a variety of otherfunctional sequences.

Also included within this disclosure are methods of sample preparationfor nucleic acid analysis, and particularly for sequencing applications.Sample preparation can generally include, e.g. obtaining a samplecomprising sample nucleic acid from a source, optionally furtherprocessing the sample, combining the sample nucleic acid with barcodedbeads, and forming emulsions containing fluidic droplets comprising thesample nucleic acid and the barcoded beads. Droplets may be generated,for example, with the aid of a microfluidic device and/or via anysuitable emulsification method. The fluidic droplets can also compriseagents capable of dissolving, degrading, or otherwise disrupting thebarcoded beads, and/or disrupting the linkage to attached sequences,thereby releasing the attached barcode sequences from the bead. Thebarcode sequences may be released either by degrading the bead,detaching the oligonucleotides from the bead such as by a cleavagereaction, or a combination of both. By amplifying (e.g., viaamplification methods described herein) the sample nucleic acid in thefluidic droplets, for example, the free barcode sequences can beattached to the sample nucleic acid. The emulsion comprising the fluidicdroplets can then be broken and, if desired, additional sequences (e.g.,sequences that aid in particular sequencing methods, additional barcodesequences, etc.) can then be added to the barcoded sample nucleic acidusing, for example, additional amplification methods. Sequencing canthen be performed on the barcoded, amplified sample nucleic acid and oneor more sequencing algorithms applied to interpret the sequencing data.As used herein, the sample nucleic acids may include any of a widevariety of nucleic acids, including, e.g., DNA and RNA, and specificallyincluding for example, genomic DNA, cDNA, mRNA total RNA, and cDNAcreated from a mRNA or total RNA transcript.

FIG. 1B illustrates an example method for barcoding and subsequentlysequencing a sample nucleic acid. First, a sample comprising nucleicacid may be obtained from a source, 111, and a set of barcoded beads maybe obtained, e.g., as described herein, 112. The beads are preferablylinked to oligonucleotides containing one or more barcode sequences, aswell as a primer, such as a random N-mer or other primer. Preferably,the barcode sequences are releasable from the barcoded beads, e.g.,through cleavage of a linkage between the barcode and the bead orthrough degradation of the underlying bead to release the barcode, or acombination of the two. For example, in certain preferred aspects, thebarcoded beads can be degraded or dissolved by an agent, such as areducing agent to release the barcode sequences. In this example, thesample comprising nucleic acid, 113, barcoded beads, 114, and e.g., areducing agent, 116, are combined and subject to partitioning. By way ofexample, such partitioning may involve introducing the components to adroplet generation system, such as a microfluidic device, 115. With theaid of the microfluidic device 115, a water-in-oil emulsion 117 may beformed, wherein the emulsion contains aqueous droplets that containsample nucleic acid, reducing agent, and barcoded beads, 117. Thereducing agent may dissolve or degrade the barcoded beads, therebyreleasing the oligonucleotides with the barcodes and random N-mers fromthe beads within the droplets, 118. The random N-mers may then primedifferent regions of the sample nucleic acid, resulting in amplifiedcopies of the sample after amplification, wherein each copy is taggedwith a barcode sequence, 119. Preferably, each droplet contains a set ofoligonucleotides that contain identical barcode sequences and differentrandom N-mer sequences. Subsequently, the emulsion is broken, 120 andadditional sequences (e.g., sequences that aid in particular sequencingmethods, additional barcodes, etc.) may be added, 122, via, for example,amplification methods (e.g., PCR). Sequencing may then be performed,123, and an algorithm applied to interpret the sequencing data, 124.Sequencing algorithms are generally capable, for example, of performinganalysis of barcodes to align sequencing reads and/or identify thesample from which a particular sequence read belongs.

The methods and compositions of this disclosure may be used with anysuitable digital processor. The digital processor may be programmed, forexample, to operate any component of a device and/or execute methodsdescribed herein. In some embodiments, bead formation may be executedwith the aid of a digital processor in communication with a dropletgenerator. The digital processor may control the speed at which dropletsare formed or control the total number of droplets that are generated.In some embodiments, attaching barcode sequences to sample nucleic acidmay be completed with the aid of a microfluidic device and a digitalprocessor in communication with the microfluidic device. In some cases,the digital processor may control the amount of sample and/or beadsprovided to the channels of the microfluidic device, the flow rates ofmaterials within the channels, and the rate at which droplets comprisingbarcode sequences and sample nucleic acid are generated.

The methods and compositions of this disclosure may be useful for avariety of different molecular biology applications including, but notlimited to, nucleic acid sequencing, protein sequencing, nucleic acidquantification, sequencing optimization, detecting gene expression,quantifying gene expression, epigenetic applications, and single-cellanalysis of genomic or expressed markers. Moreover, the methods andcompositions of this disclosure have numerous medical applicationsincluding identification, detection, diagnosis, treatment, staging of,or risk prediction of various genetic and non-genetic diseases anddisorders including cancer.

II. Partitioning of Polynucleotides

As described throughout the disclosure, certain methods, systems, andcompositions of the disclosure may utilize partitioning ofpolynucleotides into separate partitions (e.g., microwells, droplets ofan emulsion). These partitions may be used to contain polynucleotidesfor further processing, such as, for example, cutting, ligating, and/orbarcoding.

Any number of devices, systems or containers may be used to hold,support or contain partitions of polynucleotides and their fragments. Insome cases, partitions are formed from droplets, emulsions, or spots ona substrate. Weizmann et al. (Nature Methods, 2006, Vol. 3 No. 7 pages545-550). Suitable methods for forming emulsions, which can be used aspartitions or to generate microcapsules, include the methods describedin Weitz et al. (U.S. Pub. No. 2012/0211084). Partitions may also beformed through the use of wells, microwells, multi-well plates, andmicrowell arrays. Partitioning may be performed using piezoelectricdroplet generation (e.g., Bransky et al., Lab on a Chip, 2009, 9,516-520). Partitioning may be performed using surface acoustic waves(e.g., Demirci and Montesano, Lab on a Chip, 2007, 7, 1139-1145).

Each partition may also contain, or be contained within any othersuitable partition. For example, a well, microwell, hole, a surface of abead, or a tube may comprise a droplet (e.g., a droplet in an emulsion),a continuous phase in an emulsion, a spot, a capsule, or any othersuitable partition. A droplet may comprise a capsule, bead, or anotherdroplet. A capsule may comprise a droplet, bead, or another capsule.These descriptions are merely illustrative, and all suitablecombinations and pluralities are also envisioned. For example, anysuitable partition may comprise a plurality of the same or differentpartitions. In one example, a well or microwell comprises a plurality ofdroplets and a plurality of capsules. In another example, a capsulecomprises a plurality of capsules and a plurality of droplets. Allcombinations of partitions are envisioned. Table 1 shows non-limitingexamples of partitions that may be combined with each other.

TABLE 1 Examples of partitions that may be combined with each other.Well Spot Droplet Capsule Well Well inside Spot inside Droplet insideCapsule inside well well well well Spot Spot inside Spot inside Dropletinside Capsule inside well spot spot spot Droplet Droplet Droplet insideDroplet inside Droplet inside inside well spot droplet capsule Capsuleinside droplet Capsule Capsule Capsule inside Capsule inside Capsuleinside inside well spot droplet capsule Spot inside Droplet insidecapsule capsule Surface Bead inside Spot on bead Bead inside Bead insideof a well Bead inside droplet capsule Bead spot

Any partition described herein may comprise multiple partitions. Forexample, a partition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10000, or 50000 partitions. A partition may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000partitions. In some cases, a partition may comprise less than 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 partitions. In somecases, each partition may comprise 2-50, 2-20, 2-10, or 2-5 partitions.

The number of partitions employed may vary depending on the application.For example, the number of partitions may be about 5, 10, 50, 100, 250,500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000,40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000,3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The numberof partitions may be at least about 5, 10, 50, 100, 250, 500, 750, 1000,1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000,700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000,5,000,000, 10000000, 20000000, or more. The number of partitions may beless than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500,5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000,900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000,20000000. The number of partitions may be about 5-10000000, 5-5000000,5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000, 1,000-5,000,1,000-4,000, 1,000-3,000, or 1,000-2,000.

Such partitions may be pre-loaded with reagents to perform a particularreaction. For example, a capsule containing one or more reagents may beplaced within a microwell. After adding a polynucleotide sample to thewell, the capsule may be made to release its contents. The contents ofthe capsule may include, for example, restriction enzymes, ligases,barcodes, and adapters for processing the polynucleotide sample placedin the microwell.

In some cases, such partitions may be droplets of an emulsion. Forexample, a droplet of an emulsion may be an aqueous droplet in an oilphase. The droplet may comprise, for example, one or more reagents(e.g., restriction enzymes, ligases, polymerases, reagents necessary fornucleic acid amplification (e.g., primers, DNA polymerases, dNTPs,buffers)), a polynucleotide sample, and a barcode sequence. In somecases, the barcode sequence, polynucleotide sample, or any reagent maybe associated with a solid surface within a droplet. In some cases, thesolid surface is a bead. In some cases, the bead is a gel bead (seee.g., Agresti et al., U.S. Patent Publication No. 2010/0136544). In somecases the droplet is hardened into a gel bead (e.g., viapolymerization).

A species may be contained within a droplet in an emulsion containing,for example, a first phase (e.g., oil or water) forming the droplet anda second (continuous) phase (e.g., water or oil). An emulsion may be asingle emulsion, for example, a water-in-oil or an oil-in-wateremulsion. An emulsion may be a double emulsion, for example awater-in-oil-in-water or an oil-in-water-in-oil emulsion. Higher-orderemulsions are also possible. The emulsion may be held in any suitablecontainer, including any suitable partition described in thisdisclosure.

In some cases, droplets in an emulsion comprise other partitions. Adroplet in an emulsion may comprise any suitable partition including,for example, another droplet (e.g., a droplet in an emulsion), acapsule, a bead, and the like. Each partition may be present as a singlepartition or a plurality of partitions, and each partition may comprisethe same species or different species.

In one example, a droplet in an emulsion comprises a capsule comprisingreagents for sample processing. As described elsewhere in thisdisclosure, a capsule may contain one or more capsules, or otherpartitions. A sample comprising an analyte to be processed is containedwithin the droplet. A stimulus is applied to cause release of thecontents of the capsule into the droplet, resulting in contact betweenthe reagents and the analyte to be processed. The droplet is incubatedunder appropriate conditions for the processing of the analyte.Processed analyte may then be recovered. While this example describes anembodiment where a reagent is in a capsule and an analyte is in thedroplet, the opposite configuration—i.e., reagent in the droplet andanalyte in the capsule—is also possible.

The droplets in an emulsion may be of uniform size or heterogeneoussize. In some cases, the diameter of a droplet in an emulsion may beabout 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, or 1 mm. A droplet may have a diameter of at least about0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm,100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm,900 μm, or 1 mm. In some cases, a droplet may have a diameter of lessthan about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700μm, 800 μm, 900 μm, or 1 mm. In some cases, a droplet may have adiameter of about 0.001 μm to 1 mm, 0.01 μm to 900 μm, 0.1 μm to 600 μm,100 μm to 200 μm, 100 μm to 300 μm, 100 μm to 400 μm, 100 μm to 500 μm,100 μm to 600 μm, 150 μm to 200 μm, 150 μm to 300 μm, or 150 μm to 400μm.

Droplets in an emulsion also may have a particular density. In somecases, the droplets are less dense than an aqueous fluid (e.g., water);in some cases, the droplets are denser than an aqueous fluid. In somecases, the droplets are less dense than a non-aqueous fluid (e.g., oil);in some cases, the droplets are denser than a non-aqueous fluid.Droplets may have a density of about 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³,0.3 g/cm³, 0.4 g/cm³, 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Droplets mayhave a density of at least about 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³, 0.3g/cm³, 0.4 g/cm³, 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. In othercases, droplet densities may be at most about 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Such densitiescan reflect the density of the capsule in any particular fluid (e.g.,aqueous, water, oil, etc.)

Polynucleotides may be partitioned using a variety of methods. Forexample, polynucleotides may be diluted and dispensed across a pluralityof partitions. A terminal dilution of a medium comprisingpolynucleotides may be performed such that the number of partitions orwells exceeds the number of polynucleotides. The ratio of the number ofpolynucleotides to the number of partitions may range from about 0.1-10,0.5-10, 1-10, 2-10, 10-100, 100-1000, or more. The ratio of the numberof polynucleotides to the number of partitions may be about 0.1, 0.5, 1,2, 4, 8, 10, 20, 50, 100, or 1000. The ratio of the number ofpolynucleotides to the number of partitions may be at least about 0.1,0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000. The ratio of the number ofpolynucleotides to the number of partitions may be less than about 0.1,0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000.

The number of partitions employed may vary depending on the application.For example, the number of partitions may be about 5, 10, 50, 100, 250,500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, or more. Thenumber of partitions may be at least about 5, 10, 50, 100, 250, 500,750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, or more. The numberof partitions may be less than about 5, 10, 50, 100, 250, 500, 750,1000, 1500, 2000, 2500, 5000, 7500, or 10,000.

The volume of the partitions may vary depending on the application. Forexample, the volume of the partitions may be about 1000 μl, 900 μl, 800μl, 700 μl, 600 μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25μl, 10 μl, 5 μl, 1 μl, 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL,300 nL, 200 nL, 100 nL, 50 nL, 25 nL, 10 nL, or 5 nL. The volume of thepartitions may be at least about 1000 μl, 900 μl, 800 μl, 700 μl, 600μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1μl, 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100nL, 50 nL, 25 nL, 10 nL, or 5 nL. The volume of the partitions may beless than about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl, 400 μl,300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL, 800 nL,700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 25 nL, 10nL, or 5 nL.

Species may also be partitioned at a particular density. For example,species may be partitioned so that each partition contains about 1, 5,10, 50, 100, 1000, 10000, 100000, or 1000000 species per partition.Species may be partitioned so that each partition contains at leastabout 1, 5, 10, 50, 100, 1000, 10000, 100000, 1000000 or more speciesper partition. Species may be partitioned so that each partitioncontains less than about 1, 5, 10, 50, 100, 1000, 10000, 100000, or1000000 species per partition. Species may be partitioned such that eachpartition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000,10000-100000, or 100000-1000000 species per partition.

Species may be partitioned such that at least one partition comprises aspecies that is unique within that partition. This may be true for about1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of thepartitions. This may be true for at least about 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may betrue for less than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% of the partitions.

Particular polynucleotides may also be targeted to specific partitions.For example, in some cases, a capture reagent such as an oligonucleotideprobe may be immobilized in a partition to capture specificpolynucleotides through hybridization.

Polynucleotides may also be partitioned at a particular density. Forexample, polynucleotides may be partitioned such that each partitioncontains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000,10000-100000, or 100000-1000000 polynucleotides per partition.Polynucleotides may be partitioned so that each partition contains about1, 5, 10, 50, 100, 1000, 10000, 100000, 1000000 or more polynucleotidesper partition. Polynucleotides may be partitioned so that each partitioncontains less than about 1, 5, 10, 50, 100, 1000, 10000, 100000, or1000000 polynucleotides per partition. Polynucleotides may bepartitioned so that each partition contains at least about 1, 5, 10, 50,100, 1000, 10000, 100000, or 1000000 polynucleotides per partition.

Polynucleotides may be partitioned such that at least one partitioncomprises a polynucleotide sequence with a unique sequence compared toall other polynucleotide sequences contained within the same partition.This may be true for about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or more of the partitions. This may be true for less thanabout 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more ofthe partitions. This may be true for more than about 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions.

III. Beads or Particles

The methods, compositions, devices, and kits of this disclosure may beused with any suitable bead or particle, including gel beads and othertypes of beads. Beads may serve as a carrier for reagents that are to bedelivered in accordance with the methods described herein. Inparticular, these beads may provide a surface to which reagents arereleasably attached, or a volume in which reagents are entrained orotherwise releasably partitioned. These reagents may then be deliveredin accordance with a desired method, for example, in the controlleddelivery of reagents into discrete partitions. A wide variety ofdifferent reagents or reagent types may be associated with the beads,where one may desire to deliver such reagents to a partition.Non-limiting examples of such reagents include, e.g., enzymes,polypeptides, antibodies or antibody fragments, labeling reagents, e.g.,dyes, fluorophores, chromophores, etc., nucleic acids, polynucleotides,oligonucleotides, and any combination of two or more of the foregoing.In some cases, the beads may provide a surface upon which to synthesizeor attach oligonucleotide sequences. Various entities includingoligonucleotides, barcode sequences, primers, crosslinkers and the likemay be associated with the outer surface of a bead. In the case ofporous beads, an entity may be associated with both the outer and innersurfaces of a bead. The entities may be attached directly to the surfaceof a bead (e.g., via a covalent bond, ionic bond, van der Waalsinteractions, etc.), may be attached to other oligonucleotide sequencesattached to the surface of a bead (e.g. adaptor or primers), may bediffused throughout the interior of a bead and/or may be combined with abead in a partition (e.g. fluidic droplet). In preferred embodiments,the oligonucleotides are covalently attached to sites within thepolymeric matrix of the bead and are therefore present within theinterior and exterior of the bead. In some cases, an entity such as acell or nucleic acid is encapsulated within a bead. Other entitiesincluding amplification reagents (e.g., PCR reagents, primers) may alsobe diffused throughout the bead or chemically-linked within the interior(e.g., via pores, covalent attachment to polymeric matrix) of a bead.

Beads may serve to localize entities or samples. In some embodiments,entities (e.g. oligonucleotides, barcode sequences, primers,crosslinkers, adaptors and the like) may be associated with the outerand/or an inner surface of the bead. In some cases, entities may belocated throughout the bead. In some cases, the entities may beassociated with the entire surface of a bead or with at least half thesurface of the bead.

Beads may serve as a support on which to synthesize oligonucleotidesequences. In some embodiments, synthesis of an oligonucleotide maycomprise a ligation step. In some cases, synthesis of an oligonucleotidemay comprise ligating two smaller oligonucleotides together. In somecases, a primer extension or other amplification reaction may be used tosynthesize an oligonucleotide on a bead via a primer attached to thebead. In such cases, a primer attached to the bead may hybridize to aprimer binding site of an oligonucleotide that also contains a templatenucleotide sequence. The primer can then be extended by an primerextension reaction or other amplification reaction, and anoligonucleotide complementary to the template oligonucleotide canthereby be attached to the bead. In some cases, a set of identicaloligonucleotides associated with a bead may be ligated to a set ofdiverse oligonucleotides, such that each identical oligonucleotide isattached to a different member of the diverse set of oligonucleotides.In other cases, a set of diverse oligonucleotides associated with a beadmay be ligated to a set of identical oligonucleotides.

Bead Characteristics

The methods, compositions, devices, and kits of this disclosure may beused with any suitable bead. In some embodiments, a bead may be porous,non-porous, solid, semi-solid, semi-fluidic, or fluidic. In someembodiments, a bead may be dissolvable, disruptable, or degradable. Insome cases, a bead may not be degradable. In some embodiments, the beadmay be a gel bead. A gel bead may be a hydrogel bead. A gel bead may beformed from molecular precursors, such as a polymeric or monomericspecies. A semi-solid bead may be a liposomal bead. Solid beads maycomprise metals including iron oxide, gold, and silver. In some cases,the beads are silica beads. In some cases, the beads are rigid. In somecases, the beads may be flexible.

In some embodiments, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the precursors. In some cases, a precursor may be analready polymerized species capable of undergoing further polymerizationvia, for example, a chemical cross-linkage. In some cases, a precursorcomprises one or more of an acrylamide or a methacrylamide monomer,oligomer, or polymer. In some cases, the bead may comprise prepolymers,which are oligomers capable of further polymerization. For example,polyurethane beads may be prepared using prepolymers. In some cases, thebead may contain individual polymers that may be further polymerizedtogether. In some cases, beads may be generated via polymerization ofdifferent precursors, such that they comprise mixed polymers,co-polymers, and/or block co-polymers.

A bead may comprise natural and/or synthetic materials, includingnatural and synthetic polymers. Examples of natural polymers includeproteins and sugars such as deoxyribonucleic acid, rubber, cellulose,starch (e.g. amylose, amylopectin), proteins, enzymes, polysaccharides,silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) andcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some cases, a chemical cross-linker may be a precursor used tocross-link monomers during polymerization of the monomers and/or may beused to functionalize a bead with a species. In some cases, polymers maybe further polymerized with a cross-linker species or other type ofmonomer to generate a further polymeric network. Non-limiting examplesof chemical cross-linkers (also referred to as a “crosslinker” or a“crosslinker agent” herein) include cystamine, gluteraldehyde, dimethylsuberimidate, N-Hydroxysuccinimide crosslinker BS3, formaldehyde,carbodiimide (EDC), SMCC, Sulfo-SMCC, vinylsilance,N,N′diallyltartardiamide (DATD), N,N′-Bis(acryloyl)cystamine (BAC), orhomologs thereof. In some cases, the crosslinker used in the presentdisclosure contains cystamine.

Crosslinking may be permanent or reversible, depending upon theparticular crosslinker used. Reversible crosslinking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine. In some embodiments, disulfide linkages may be formed betweenmolecular precursor units (e.g. monomers, oligomers, or linearpolymers). In some embodiments, disulfide linkages may be may be formedbetween molecular precursor units (e.g. monomers, oligomers, or linearpolymers) or precursors incorporated into a bead and oligonucleotides.

Cystamine (including modified cystamines), for example, is an organicagent comprising a disulfide bond that may be used as a crosslinkeragent between individual monomeric or polymeric precursors of a bead.Polyacrylamide may be polymerized in the presence of cystamine or aspecies comprising cystamine (e.g., a modified cystamine) to generatepolyacrylamide gel beads comprising disulfide linkages (e.g., chemicallydegradable beads comprising chemically-reducible cross-linkers). Thedisulfide linkages may permit the bead to be degraded (or dissolved)upon exposure of the bead to a reducing agent.

In at least one alternative example, chitosan, a linear polysaccharidepolymer, may be crosslinked with glutaraldehyde via hydrophilic chainsto form a bead. Crosslinking of chitosan polymers may be achieved bychemical reactions that are initiated by heat, pressure, change in pH,and/or radiation.

In some embodiments, the bead may comprise covalent or ionic bondsbetween polymeric precursors (e.g. monomers, oligomers, linearpolymers), oligonucleotides, primers, and other entities. In some cases,the covalent bonds comprise carbon-carbon bonds or thioether bonds.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more species (e.g., barcodesequence, primer, other oligonucleotide) to the bead. In some cases, anacrydite moiety can refer to an acrydite analogue generated from thereaction of acrydite with one or more species, such as, for example, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as an oligonucleotide(e.g., barcode sequence, primer, other oligonucleotide). For example,acrydite moieties may be modified with thiol groups capable of forminga, disulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment is reversible, such that when the disulfide bond is broken(e.g., in the presence of a reducing agent), the agent is released fromthe bead. In other cases, an acrydite moiety comprises a reactivehydroxyl group that may be used for attachment.

Functionalization of beads for attachment of other species, e.g.,nucleic acids, may be achieved through a wide range of differentapproaches, including activation of chemical groups within a polymer,incorporation of active or activatable functional groups in the polymerstructure, or attachment at the pre-polymer or monomer stage in beadproduction.

For example, in some examples, precursors (e.g., monomers,cross-linkers) that are polymerized to form a bead may comprise acryditemoieties, such that when a bead is generated, the bead also comprisesacrydite moieties. Often, the acrydite moieties are attached to anoligonucleotide sequence, such as a primer (e.g., a primer for one ormore of amplifying target nucleic acids and/or sequencing target nucleicacids barcode sequence, binding sequence, or the like)) that is desiredto be incorporated into the bead. In some cases, the primer comprises aP5 sequence. For example, acrylamide precursors (e.g., cross-linkers,monomers) may comprise acrydite moieties such that when they arepolymerized to form a bead, the bead also comprises acrydite moieties.

In some cases, precursors such as monomers and cross-linkers maycomprise, for example, a single oligonucleotide (e.g., such as a primeror other sequence) or other species. FIG. 29A depicts an example monomercomprising an acrydite moiety and single P5 sequence linked to theacrydite moiety via a disulfide bond. In some cases, precursors such asmonomers and cross-linkers may comprise multiple oligonucleotides, othersequences, or other species. FIG. 29B depicts an example monomercomprising multiple acrydite moieties each linked to a P5 primer via adisulfide bond. Moreover, FIG. 29C depicts an example cross-linkercomprising multiple acrydite moieties each linked to a P5 species via adisulfide bond. The inclusion of multiple acrydite moieties or otherlinker species in each precursor may improve loading of a linked species(e.g., an oligonucleotide) into beads generated from the precursorsbecause each precursor can comprise multiple copies of a species to beloaded.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group, as shown in FIG. 31. In some cases, acrylic acid (aspecies comprising free COOH groups), acrylamide, andbis(acryloyl)cystamine can be co-polymerized together to generate a gelbead comprising free COOH groups. The COOH groups of the gel bead can beactivated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)and N-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM) as shown in FIG. 31) such that they are reactive (e.g., reactiveto amine functional groups where EDC/NHS or DMTMM are used foractivation). The activated COOH groups can then react with anappropriate species (e.g., a species comprising an amine functionalgroup where the carboxylic acid groups are activated to be reactive withan amine functional group) comprising a moiety to be linked to the bead.

An example species comprising an amine group linked to a P5 primer via adisulfide bond (e.g., H₂N—C₆—S—S—C₆—P₅) is shown in FIG. 31. COOHfunctional groups of a gel bead can be activated with EDC/NHS or DMTMMto generate an amine reactive species at one or more of the COOH sites.The amine group of the species H₂N—C₆—S—S—C₆—P₅ moiety can then reactwith the activated carboxylic acid such that the moiety and attached P5oligonucleotide becomes covalently linked to the bead as shown in FIG.31. Unreacted COOH species can be converted to other species such thatthey are blocked.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange)) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, though, free thiols of thebeads may react with any other suitable group. For example, free thiolsof the beads may react with species comprising an acrydite moiety. Thefree thiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such as, for example,N-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than about 10000, 100000, 1000000, 10000000, 100000000,1000000000, 10000000000, or 100000000000) of reducing agent may be usedfor reduction. Controlling the number of disulfide linkages that arereduced to free thiols may be useful in ensuring bead structuralintegrity during functionalization. In some cases, optically-activeagents, such as fluorescent dyes may be may be coupled to beads via freethiol groups of the beads and used to quantify the number of free thiolspresent in a bead and/or track a bead.

An example scheme for functionalizing gel beads comprising disulfidelinkages is shown in FIG. 35A. As shown, beads 3501 (e.g., gel beads)comprising disulfide linkages can be generated using, for example, anyof the methods described herein. Upon action of a reducing agent 3502(e.g., DTT, TCEP, or any other reducing agent described herein) at aconcentration not suitable for bead degradation, some of the gel bead3501 disulfide linkages can be reduced to free thiols to generate beads3503 comprising free thiol groups. Upon removal of the reducing agent(e.g., via washing) 3504, beads 3503 can be reacted with anacrydite-S—S-species moiety 3505 comprising a species to be loaded(e.g., P5 oligonucleotide shown, but the species may be another type ofpolynucleotide such as, for, example, an oligonucleotide comprising P5,a barcode sequence, R1, and a random N-mer) linked to the acrydite via adisulfide bond. Moiety 3505 can couple with the gel beads 3503 viaMichael addition chemistry to generate beads 3506 comprising moiety3505. The generated beads 3506 can then be purified (e.g., via washing)by removing unwanted (e.g., non-attached) species.

Another example scheme for functionalizing gel beads comprisingdisulfide linkages is shown in FIG. 35B. As shown, beads 3501 (e.g., gelbeads) comprising disulfide linkages can be generated using, forexample, any of the methods described herein. Upon action of a reducingagent 3502 (e.g., DTT, TCEP, or any other reducing agent describedherein) at a concentration not suitable for bead degradation, some ofthe gel beads 3501 disulfide linkages can be reduced to free thiols togenerate beads 3503 comprising free thiol groups. Upon removal of thereducing agent (e.g., via washing) 3504, beads 3503 can be reacted with2,2′-Dithiopyridine 3507 to generate gel beads 3509 linked to a pyridinemoiety via a disulfide bond. As an alternative to 2,2′-Dithiopyridine,other similar species, such as 4,4′-Dithiopyridine or5,5′-dithiobis-(2-nitrobenzoic acid) (e.g., DTNB or Ellman's Reagent)may be used. 2,2′-Dithiopyridine 3507 can couple with the gel beads 3503via disulfide exchange to generate beads 3509 comprising a pyridinemoiety linked to the beads 3509 via a disulfide bond. Gel beads 3509 canthen be separated from unreacted species (e.g., via washing).

The purified gel beads 3509 can then be reacted with a moiety 3508comprising a species of interest (e.g., a P5 oligonucleotide as shown)to be coupled to the gel beads and a free thiol group. In some cases,moiety 3508 may be generated from another species comprising a disulfidebond, such that when the disulfide bond is reduced (e.g., via the actionof a reducing agent such as DTT, TCEP, etc.), moiety 3508 with a freethiol group is obtained. Moiety 3508 can participate in thiol-disulfideexchange with the pyridine group of beads 3509 to generate gel beads3510 comprising moiety 3508. The pyridine group is generally a goodleaving group, which can permit effective thiol-disulfide exchange withthe free thiol of moiety 3508. The generated beads 3510 can then bepurified (e.g., via washing) by removing unwanted species.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of a species aftergel bead formation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid in species(e.g., a primer, a P5 primer) infiltration into the bead duringsubsequent functionalization of the bead with the species.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Also, species loading may beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

In some cases, acrydite moieties linked to precursors, another specieslinked to a precursor, or a precursor itself comprise a labile bond,such as, for example, chemically, thermally, or photo-sensitive bondse.g., disulfide bonds, UV sensitive bonds, or the like. Once acryditemoieties or other moieties comprising a labile bond are incorporatedinto a bead, the bead may also comprise the labile bond. The labile bondmay be, for example, useful in reversibly linking (e.g., covalentlylinking) species (e.g., barcodes, primers, etc.) to a bead. In somecases, a thermally labile bond may include a nucleic acid hybridizationbased attachment, e.g., where an oligonucleotide is hybridized to acomplementary sequence that is attached to the bead, such that thermalmelting of the hybrid releases the oligonucleotide, e.g., a barcodecontaining sequence, from the bead or microcapsule. Moreover, theaddition of multiple types of labile bonds to a gel bead may result inthe generation of a bead capable of responding to varied stimuli. Eachtype of labile bond may be sensitive to an associated stimulus (e.g.,chemical stimulus, light, temperature, etc.) such that release ofspecies attached to a bead via each labile bond may be controlled by theapplication of the appropriate stimulus. Such functionality may beuseful in controlled release of species from a gel bead. In some cases,another species comprising a labile bond may be linked to a gel beadafter gel bead formation via, for example, an activated functional groupof the gel bead as described above. As will be appreciated, barcodesthat are releasably, cleavably or reversibly attached to the beadsdescribed herein include barcodes that are released or releasablethrough cleavage of a linkage between the barcode molecule and the bead,or that are released through degradation of the underlying bead itself,allowing the barcodes to be accessed or accessible by other reagents, orboth. In general, the barcodes that are releasable as described herein,may generally be referred to as being activatable, in that they areavailable for reaction once released. Thus, for example, an activatablebarcode may be activated by releasing the barcode from a bead (or othersuitable type of partition described herein). As will be appreciated,other activatable configurations are also envisioned in the context ofthe described methods and systems. In particular, reagents may beprovided releasably attached to beads, or otherwise disposed inpartitions, with associated activatable groups, such that once deliveredto the desired set of reagents, e.g., through co-partitioning, theactivatable group may be reacted with the desired reagents. Suchactivatable groups include caging groups, removable blocking orprotecting groups, e.g., photolabile groups, heat labile groups, orchemically removable groups.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). 1001581A bead may be linked to a varied number ofacrydite moieties. For example, a bead may comprise about 1, 10, 100,1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000, or10000000000 acrydite moieties linked to the beads. In other examples, abead may comprise at least 1, 10, 100, 1000, 10000, 100000, 1000000,10000000, 100000000, 1000000000, or 10000000000 acrydite moieties linkedto the beads. For example, a bead may comprise about 1, 10, 100, 1000,10000, 100000, 1000000, 10000000, 100000000, 1000000000, or 10000000000oligonucleotides covalently linked to the beads, such as via an acryditemoiety. In other examples, a bead may comprise at least 1, 10, 100,1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000, or10000000000 oligonucleotides covalently linked to the beads, such as viaan acrydite moiety.

Species that do not participate in polymerization may also beencapsulated in beads during bead generation (e.g., duringpolymerization of precursors). Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,oligonucleotides, species necessary for a nucleic acid amplificationreaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionicco-factors)) including those described herein, species necessary forenzymatic reactions (e.g., enzymes, co-factors, substrates), or speciesnecessary for a nucleic acid modification reaction such aspolymerization, ligation, or digestion. Trapping of such species may becontrolled by the polymer network density generated duringpolymerization of precursors, control of ionic charge within the gelbead (e.g., via ionic species linked to polymerized species), or by therelease of other species. Encapsulated species may be released from abead upon bead degradation and/or by application of a stimulus capableof releasing the species from the bead.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be about 1 μm, 5 μm, 100 μm, 250 μm, 500 μm, or 1mm. In some cases, a bead may have a diameter of at least about 1 μm, 5μm, 100 μm, 250 μm, 500 μm, 1 mm, or more. In some cases, a bead mayhave a diameter of less than about 1 mm, 5 μm, 10 μm, 20 μm, 30 μm, 40μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 250μm, 500 μm, or 1 mm. In some cases, a bead may have a diameter in therange of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

In certain preferred aspects, the beads are provided as a population ofbeads having a relatively monodisperse size distribution. As will beappreciated, in some applications, where it is desirable to providerelatively consistent amounts of reagents within partitions, maintainingrelatively consistent bead characteristics, such as size, contributes tothat overall consistency. In particular, the beads described herein mayhave size distributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, oreven less than 5%.

Beads may be of a regular shape or an irregular shape. Examples of beadshapes include spherical, non-spherical, oval, oblong, amorphous,circular, cylindrical, and homologs thereof.

Degradable Beads

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, e.g., barcode containingoligonucleotides, described above, the beads may be degradable,disruptable, or dissolvable spontaneously or upon exposure to one ormore stimuli (e.g., temperature changes, pH changes, exposure toparticular chemical species or phase, exposure to light, reducing agent,etc.). In some cases, a bead may be dissolvable, such that materialcomponents of the beads are solubilized when exposed to a particularchemical species or an environmental changes, such as, for example,temperature, or pH. For example, a gel bead may be degraded or dissolvedat elevated temperature and/or in basic conditions. In some cases, abead may be thermally degradable such that when the bead is exposed toan appropriate change in temperature (e.g., heat), the bead degrades.Degradation or dissolution of a bead bound to a species (e.g., a nucleicacid species) may result in release of the species from the bead.

A degradable bead may comprise one or more species with a labile bondsuch that when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond is broken and the bead is degraded. Forexample, a polyacrylamide gel bead may comprise cystamine crosslinkers.Upon exposure of the bead to a reducing agent, the disulfide bonds ofthe cystamine are broken and the bead is degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., an oligonucleotide, a barcode sequence) from the beadwhen the appropriate stimulus is applied to the bead. For example, for aspecies bound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species are released within the dropletwhen the appropriate stimulus is applied. The free species may interactwith other species. For example, a polyacrylamide bead comprisingcystamine and linked, via a disulfide bond, to a barcode sequence, maybe combined with a reducing agent within a droplet of a water-in-oilemulsion. Within the droplet, the reducing agent breaks the variousdisulfide bonds resulting in bead degradation and release of the barcodesequence into the aqueous, inner environment of the droplet. In anotherexample, heating of a droplet comprising a bead-bound barcode sequencein basic solution may also result in bead degradation and release of theattached barcode sequence into the aqueous, inner environment of thedroplet.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

As will be appreciated, where degradable beads are provided, it may bedesirable to avoid exposing such beads to the stimulus or stimuli thatcause such degradation prior to the desired time, in order to avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics, clumping andaggregation. By way of example, where beads comprise reduciblecross-linking groups, such as disulfide groups, it will be desirable toavoid contacting such beads with reducing agents, e.g., DTT or otherdisulfide cleaving reagents. In such cases, treatments to the beadsdescribed herein will, in some cases be provided to be free of reducingagents, such as DTT. Because reducing agents are often provided incommercial enzyme preparations, it is often desirable to providereducing agent free (or DTT free) enzyme preparations in treating thebeads described herein. Examples of such enzymes include, e.g.,polymerase enzyme preparations, ligase enzyme preparations, as well asmany other enzyme preparations that may be used to treat the beadsdescribed herein. By “reducing agent free” or “DTT free” preparationsmeans that the preparation will have less than 1/10th, less than1/50^(th), and even less than 1/100^(th) of the lower ranges for suchmaterials used in degrading the beads. For example, for DTT, thereducing agent free preparation will typically have less than 0.01 mM,0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than 0.0001 mM DTTor less. In many cases, the amount of DTT will be undetectable.

Methods for Degrading Beads

In some cases, a stimulus may be used to trigger degrading of the bead,which may result in the release of contents from the bead. Generally, astimulus may cause degradation of the bead structure, such asdegradation of the covalent bonds or other types of physicalinteraction. These stimuli may be useful in inducing a bead to degradeand/or to release its contents. Examples of stimuli that may be usedinclude chemical stimuli, thermal stimuli, light stimuli and anycombination thereof, as described more fully below.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degrading the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

The methods, compositions, devices, and kits of this disclosure may beused with any suitable agent to degrade beads. In some embodiments,changes in temperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at 0.1 mM, 0.5 mM, 1 mM, 5mM, or 10 mM. The reducing agent may be present at more than 0.1 mM, 0.5mM, 1 mM, 5 mM, 10 mM, or more. The reducing agent may be present atless than 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.

Timing of Degrading Step

Beads may be degraded to release contents attached to and containedwithin the bead. This degrading step may occur simultaneously as thesample is combined with the bead. This degrading step may occursimultaneously when the sample is combined with the bead within afluidic droplet that may be formed in a microfluidic device. Thisdegrading step may occur after the sample is combined with the beadwithin a fluidic droplet that may be formed in a microfluidic device. Aswill be appreciated, in many applications, the degrading step may notoccur.

The reducing agent may be combined with the sample and then with thebead. In some cases, the reducing agent may be introduced to amicrofluidic device as the same time as the sample. In some cases, thereducing agent may be introduced to a microfluidic device after thesample is introduced. In some cases, the sample may be mixed with thereducing agent in a microfluidic device and then contacted with the gelbead in the microfluidic device. In some embodiments, the sample may bepre-mixed with the reducing agent and then added to the device andcontacted with the gel bead.

A degradable bead may degrade instantaneously upon application of theappropriate stimuli. In other cases, degradation of the bead may occurover time. For example, a bead may degrade upon application of anappropriate stimulus instantaneously or within about 0, 0.01, 0.1, 0.5,1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 11, 12, 13, 14, 15 or 20 minutes. In otherexamples, a bead may degrade upon application of a proper stimulusinstantaneously or within at most about 0, 0.01, 0.1, 0.5, 1, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, 10.0, 11, 12, 13, 14, 15 or 20 minutes.

Beads may also be degraded at different times, relative to combiningwith a sample. For example, the bead may be combined with the sample andsubsequently degraded at a point later in time. The time betweencombining the sample with the bead and subsequently degrading the beadmay be about 0.0001, 0.001, 0.01, 1, 10, 30, 60, 300, 600, 1800, 3600,18000, 36000, 86400, 172800, 432000, or 864000 seconds. The time betweencombining the sample with the bead and subsequently degrading the beadmay be more than about 0.0001, 0.001, 0.01, 1, 10, 30, 60, 300, 600,1800, 3600, 18000, 36000, 86400, 172800, 432000, 864000 seconds or more.The time between combining the sample with the bead and subsequentlydegrading the bead may be less than about 0.0001, 0.001, 0.01, 1, 10,30, 60, 300, 600, 1800, 3600, 18000, 36000, 86400, 172800, 432000, or864000 seconds.

Preparing Beads Pre-Functionalized with Oligonucleotides

The beads described herein may be produced using a variety of methods.In some cases, beads may be formed from a liquid containing molecularprecursors (e.g. linear polymers, monomers, cross-linkers). The liquidis then subjected to a polymerization reaction, and thereby hardens orgels into a bead (or gel bead). The liquid may also contain entitiessuch as oligonucleotides that become incorporated into the bead duringpolymerization. This incorporation may be via covalent or non-covalentassociation with the bead. For example, in some cases, theoligonucleotides may be entrained within a bead during formation.Alternatively, they may be coupled to the bead or the bead frameworkeither during formation or following formation. Often, theoligonucleotides are connected to an acrydite moiety that becomescross-linked to the bead during the polymerization process. In somecases, the oligonucleotides are attached to the acrydite moiety by adisulfide linkage. As a result, a composition comprising abead-acrydite-S—S-oligonucleotide linkage is formed. FIG. 4A is anexemplary diagram of a bead functionalized with an acrydite-linkedprimer.

In one exemplary process, functionalized beads may be generated bymixing a plurality of polymers and/or monomers with one or moreoligonucleotides, such as, for example, one or more oligonucleotidesthat comprises a primer (e.g., a universal primer, a sequencing primer).The polymers and/or monomers may comprise acrylamide and may becrosslinked such that disulfide bonds form between the polymers and/ormonomers, resulting in the formation of hardened beads. Theoligonucleotides may be covalently linked to the plurality of polymersand/or monomers during the formation of the hardened beads (e.g.,contemporaneously) or may be covalently linked to the plurality ofpolymers and/or monomers after the formation of the hardened beads(e.g., sequentially). In some cases, the oligonucleotides may be linkedto the beads via an acrydite moiety.

In most cases, a population of beads is pre-functionalized with theidentical oligonucleotide such as a universal primer or primer bindingsite. In some cases, the beads in a population of beads arepre-functionalized with multiple different oligonucleotides. Theseoligonucleotides may optionally include any of a variety of differentfunctional sequences, e.g., for use in subsequent processing orapplication of the beads. Functional sequences may include, e.g., primersequences, such as targeted primer sequences, universal primersequences, e.g., primer sequences that are sufficiently short to be ableto hybridize to and prime extension from large numbers of differentlocations on a sample nucleic acid, or random primer sequences,attachment or immobilization sequences, ligation sequences, hairpinsequences, tagging sequences, e.g., barcodes or sample index sequences,or any of a variety of other nucleotide sequences.

By way of example, in some cases, the universal primer (e.g., P5 orother suitable primer) may be used as a primer on each bead, to attachadditional content (e.g., barcodes, random N-mers, other functionalsequences) to the bead. In some cases, the universal primer (e.g., P5)may also be compatible with a sequencing device, and may later enableattachment of a desired strand to a flow cell within the sequencingdevice. For example, such attachment or immobilization sequences mayprovide a complementary sequence to oligonucleotides that are tetheredto the surface of a flow cell in a sequencing device, to allowimmobilization of the sequences to that surface for sequencing.Alternatively, such attachments sequences may additionally be providedwithin, or added to the oligonucleotide sequences attached to the beads.In some cases, the beads and their attached species may be provided tobe compatible with subsequent analytical process, such as sequencingdevices or systems. In some cases, more than one primer may be attachedto a bead and more than one primer may contain a universal sequence, inorder to, for example, allow for differential processing of theoligonucleotide as well as any additional sequences that are coupled tothat sequence, in different sequential or parallel processing steps,e.g., a first primer for amplification of a target sequence, with asecond primer for sequencing the amplified product. For example, in somecases, the oligonucleotides attached to the beads will comprise a firstprimer sequence for conducting a first amplification or replicationprocess, e.g., extending the primer along a target nucleic acidsequence, in order to generate an amplified barcoded target sequence(s).By also including a sequencing primer within the oligonucleotides, theresulting amplified target sequences will include such primers, and bereadily transferred to a sequencing system. For example, in some cases,e.g., where one wishes to sequence the amplified targets using, e.g., anIllumina sequencing system, an R1 primer or primer binding site may alsobe attached to the bead.

Entities incorporated into the beads may include oligonucleotides havingany of a variety of functional sequences as described above. Forexample, these oligonucleotides may include any one or more of P5, R1,and R2 sequences, non cleavable 5′acrydite-P5, a cleavable 5′acrydite-SS-P5, R1c, sequencing primer, read primer, universal primer,P5_U, a universal read primer, and/or binding sites for any of theseprimers. In some cases, a primer may contain one or more modifiednucleotides nucleotide analogues, or nucleotide mimics. For example, insome cases, the oligonucleotides may include peptide nucleic acids(PNAs), locked nucleic acid (LNA) nucleotides, or the like. In somecases, these oligonucleotides may additionally or alternatively includenucleotides or analogues that may be processed differently, in order toallow differential processing at different steps of their application.For example, in some cases one or more of the functional sequences mayinclude a nucleotide or analogue that is not processed by a particularpolymerase enzyme, thus being uncopied in a process step utilizing thatenzyme. For example, e.g., in some cases, one or more of the functionalsequence components of the oligonucleotides will include, e.g., a uracilcontaining nucleotide, a nucleotide containing a non-native base, ablocker oligonucleotide, a blocked 3′ end, 3′ddCTP. FIG. 19 providesadditional examples. As will be appreciated, sequences of any of theseentities may function as primers or primer binding sites depending onthe particular application.

Polymerization may occur spontaneously. In some cases, polymerizationmay be initiated by an initiator and/or an accelerator, byelectromagnetic radiation, by temperature changes (e.g., addition orremoval of heat), by pH changes, by other methods, and combinationsthereof. An initiator may refer to a species capable of initiating apolymerization reaction by activating (e.g., via the generation of freeradicals) one or more precursors used in the polymerization reaction. Anaccelerator may refer to a species capable of accelerating the rate atwhich a polymerization reaction occurs. In some cases, an acceleratormay speed up the activation of an initiator (e.g., via the generation offree radicals) used to then activate monomers (e.g., via the generationof free radicals) and, thus, initiate a polymerization reaction. In somecases, faster activation of an initiator can give rise to fasterpolymerization rates. In some cases, though, acceleration may also beachieved via non-chemical means such as thermal (e.g., addition andremoval of heat) means, various types of radiative means (e.g., visiblelight, UV light, etc.), or any other suitable means. To create dropletscontaining molecular precursors, which may then polymerize to formhardened beads, an emulsion technique may be employed. For example,molecular precursors may be added to an aqueous solution. The aqueoussolution may then be emulsified with an oil (e.g., by agitation,microfluidic droplet generator, or other method). The molecularprecursors may then be polymerized in the emulsified droplets to formthe beads.

An emulsion may be prepared, for example, by any suitable method,including methods known in the art, such as bulk shaking, bulkagitation, flow focusing, and microsieve (See e.g., Weizmann et al.,Nature Methods, 2006, 3(7):545-550; Weitz et al. U.S. Pub. No.2012/0211084). In some cases, an emulsion may be prepared using amicrofluidic device. In some cases, water-in-oil emulsions may be used.These emulsions may incorporate fluorosurfactants such as Krytox FSHwith a PEG-containing compound such as bis krytox peg (BKP). In somecases, oil-in-water emulsions may be used. In some cases, polydisperseemulsions may be formed. In some cases, monodisperse emulsions may beformed. In some cases, monodisperse emulsions may be formed in amicrofluidic flow focusing device. (Gartecki et al., Applied PhysicsLetters, 2004, 85(13):2649-2651).

In at least one example, a microfluidic device for making the beads maycontain channel segments that intersect at a single cross intersectionthat combines two or more streams of immiscible fluids, such as anaqueous solution containing molecular precursors and an oil. Combiningtwo immiscible fluids at a single cross intersection may cause fluidicdroplets to form. The size of the fluidic droplets formed may dependupon the flow rate of the fluid streams entering the fluidic cross, theproperties of the two fluids, and the size of the microfluidic channels.Initiating polymerization after formation of fluidic droplets exitingthe fluidic cross may cause hardened beads to form from the fluidicdroplets. Examples of microfluidic devices, channel networks and systemsfor generating droplets, both for bead formation and for partitioningbeads into discrete droplets as discussed elsewhere herein, aredescribed for example in U.S. Provisional Patent Application No.61/977,804, filed Apr. 4, 2014, and incorporated herein by reference inits entirety for all purposes.

To manipulate when individual molecular precursors, oligomers, orpolymers begin to polymerize to form a hardened bead, an initiatorand/or accelerator may be added at different points in the beadformation process. An accelerator may be an agent which may initiate thepolymerization process (e.g., in some cases, via activation of apolymerization initiator) and thus may reduce the time for a bead toharden. In some cases, a single accelerator or a plurality ofaccelerators may be used for polymerization. Careful tuning ofacceleration can be important in achieving suitable polymerizationreactions. For example, if acceleration is too fast, weight andexcessive chain transfer events may cause poor gel structure and lowloading of any desired species. If acceleration is too slow, highmolecular weight polymers can generate trapped activation sites (e.g.,free radicals) due to polymer entanglement and high viscosities. Highviscosities can impede diffusion of species intended for bead loading,resulting in low to no loading of the species. Tuning of acceleratoraction can be achieved, for example, by selecting an appropriateaccelerator, an appropriate combination of accelerators, or by selectingthe appropriate accelerator(s) and any stimulus (e.g., heat,electromagnetic radiation (e.g., light, UV light), another chemicalspecies, etc.) capable of modulating accelerator action. Tuning ofinitiator action may also be achieved in analogous fashion.

An accelerator may be water-soluble, oil-soluble, or may be bothwater-soluble and oil-soluble. For example, an accelerator may betetramethylethylenediamine (TMEDA or TEMED), dimethylethylenediamine,N,N, N,′N′-tetramethylmethanediamine, N,N′-dimorpholinomethane, orN,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine. For example, aninitiator may be ammonium persulfate (APS), calcium ions, or any of thecompounds (I-IX) shown in FIG. 32. The compounds (I-IX) shown in FIG. 32can function as water-soluble azo-based initiators. Azo-based initiatorsmay be used in the absence of TEMED and APS and can function as thermalbased initiators. A thermal based initiator can activate species (e.g.,via the generation of free radicals) thermally and, thus, the rate ofinitiator action can be tuned by temperature and/or the concentration ofthe initiator. A polymerization accelerator or initiator may includefunctional groups including phosphonate, sulfonate, carboxylate,hydroxyl, albumin binding moieties, N-vinyl groups, and phospholipids. Apolymerization accelerator or initiator may be a low molecular weightmonomeric-compound. An accelerator or initiator may be a) added to theoil prior to droplet generation, b) added in the line after dropletgeneration, c) added to the outlet reservoir after droplet generation,or d) combinations thereof.

Polymerization may also be initiated by electromagnetic radiation.Certain types of monomers, oligomers, or polymers may containlight-sensitive properties. Thus, polymerization may be initiated byexposing such monomers, oligomers, or polymers to UV light, visiblelight, UV light combined with a sensitizer, visible light combined witha sensitizer, or combinations thereof. An example of a sensitizer may beriboflavin.

The time for a bead to completely polymerize or harden may varydepending on the size of the bead, whether an accelerator may be added,when an accelerator may be added, the type of initiator, whenelectromagnetic radiation may be applied, the temperature of solution,the polymer composition, the polymer concentration, and other relevantparameters. For example, polymerization may be complete after about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.Polymerization may be complete after more than about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes or more. Polymerizationmay be complete in less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 minutes.

Beads may be recovered from emulsions (e.g. gel-water-oil) by continuousphase exchange. Excess aqueous fluid may be added to the emulsion (e.g.gel-water-oil) and the hardened beads may be subjected to sedimentation,wherein the beads may be aggregated and the supernatant containingexcess oil may be removed. This process of adding excess aqueous fluidfollowed by sedimentation and removal of excess oil may be repeateduntil beads are suspended in a given purity of aqueous buffer, withrespect to the continuous phase oil. The purity of aqueous buffer may beabout 80%, 90%, 95%, 96%, 97%, 98%, or 99% (v/v). The purity of aqueousbuffer may be more than about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more(v/v). The purity of aqueous buffer may be less than about 80%, 90%,95%, 96%, 97%, 98%, or 99% (v/v). The sedimentation step may be repeatedabout 2, 3, 4, or 5 times. The sedimentation step may be repeated morethan about 2, 3, 4, 5 times or more. The sedimentation step may berepeated less than about 2, 3, 4, or 5 times. In some cases,sedimentation and removal of the supernatant may also remove un-reactedstarting materials.

Examples of droplet generators may include single flow focuser, parallelflow focuser, and microsieve membrane, such as those used by Nanomi B.V., and others. Preferably, a microfluidic device is used to generatethe droplets.

An example emulsion based scheme for generating gel beadspre-functionalized with an acrydite moiety linked to a P5 primer via adisulfide bond is depicted in FIGS. 30A-30C. As shown in FIG. 30A,acrylamide, bis(acryloyl)cystamine, acrydite-S—S-P5 moieties, andammonium persulfate are combined into a droplets of an emulsion. TEMEDcan be added to the emulsion oil phase and can diffuse into the dropletsto initiate the polymerization reaction. As shown in FIG. 30A, TEMEDaction on ammonium persulfate results in the generation of SO₄ ⁻ freeradicals that can then activate the carbon-carbon double bond of theacrylamide via generation of a free radical at one of the carbons of thecarbon-carbon double bond.

As shown in FIG. 30B, activated acrylamide can react with non-activatedacrylamide (again, at its carbon-carbon double bond) to beginpolymerization. Each product generated can again be activated via theformation of a free radical resulting in polymer propagation. Moreover,both the bis(acryloyl)cystamine cross-linker and acrydite-S—S-P5moieties comprise carbon-carbon double bonds that can react withactivated species and the products themselves can then become activated.The inclusion of the bis(acryloyl)cystamine cross-linker into thepolymerization reaction can result in cross-linking of polymer chainsthat are generated as shown in FIG. 30C. Thus, a hydrogel polymernetwork comprising acrydite-S—S-P5 moieties linked to polymer backbonescan be generated, as depicted in FIG. 30C. The polymerization reactioncan continue until it terminates. Upon reaction termination, continuousphase exchange or other suitable method can be used to break theemulsion and obtain gel beads comprising a cross-linked hydrogel (shownschematically in FIG. 30A) coupled to the acrydite-S—S-P5 moieties.

Barcode and Random N-mers (Introduction)

Certain applications, for example polynucleotide sequencing, may rely onunique identifiers (“barcodes”) to identify a sequence and, for example,to assemble a larger sequence from sequenced fragments. Therefore, itmay be desirable to add barcodes to polynucleotide fragments beforesequencing. In the case of nucleic acid applications, such barcodes aretypically comprised of a relatively short sequence of nucleotidesattached to a sample sequence, where the barcode sequence is eitherknown, or identifiable by its location or sequence elements. In somecases, a unique identifier may be useful for sample indexing. In somecases, though, barcodes may also be useful in other contexts. Forexample, a barcode may serve to track samples throughout processing(e.g., location of sample in a lab, location of sample in plurality ofreaction vessels, etc.); provide manufacturing information; trackbarcode performance over time (e.g., from barcode manufacturing to use)and in the field; track barcode lot performance over time in the field;provide product information during sequencing and perhaps triggerautomated protocols (e.g., automated protocols initiated and executedwith the aid of a computer) when a barcode associated with the productis read during sequencing; track and troubleshoot problematic barcodesequences or product lots; serve as a molecular trigger in a reactioninvolving the barcode, and combinations thereof. In particularlypreferred aspects, and as alluded to above, barcode sequence segments asdescribed herein, can be used to provide linkage information as betweentwo discrete determined nucleic acid sequences. This linkage informationmay include, for example, linkage to a common sample, a common reactionvessel, e.g., a well or partition, or even a common starting nucleicacid molecule. In particular, by attaching common barcodes to a specificsample component, or subset of sample components within a given reactionvolume, one can attribute the resulting sequences bearing that barcodeto that reaction volume. In turn, where the sample is allocated to thatreaction volume based upon its sample of origin, the processing steps towhich it is subsequently exposed, or on an individual molecule basis,one can better identify the resulting sequences as having originatedfrom that reaction volume.

Barcodes may be generated from a variety of different formats, includingbulk synthesized polynucleotide barcodes, randomly synthesized barcodesequences, microarray based barcode synthesis, native nucleotides,partial complement with N-mer, random N-mer, pseudo random N-mer, orcombinations thereof. Synthesis of barcodes is described herein, as wellas in, for example, in U.S. patent application Ser. No. 14/175,973,filed Feb. 7, 2014, the full disclosure of which is hereby incorporatedherein by reference in its entirety for all purposes.

As described above, oligonucleotides incorporating barcode sequencesegments, which function as a unique identifier, may also includeadditional sequence segments. Such additional sequence segments mayinclude functional sequences, such as primer sequences, primer annealingsite sequences, immobilization sequences, or other recognition orbinding sequences useful for subsequent processing, e.g., a sequencingprimer or primer binding site for use in sequencing of samples to whichthe barcode containing oligonucleotide is attached. Further, as usedherein, the reference to specific functional sequences as being includedwithin the barcode containing sequences also envisioned the inclusion ofthe complements to any such sequences, such that upon complementaryreplication will yield the specific described sequence.

In some examples, barcodes or partial barcodes may be generated fromoligonucleotides obtained from or suitable for use in an oligonucleotidearray, such as a microarray or bead array. In such cases,oligonucleotides of a microarray may be cleaved, (e.g., using cleavablelinkages or moieties that anchor the oligonucleotides to the array (suchas photoclevable, chemically cleavable, or otherwise cleavablelinkages)) such that the free oligonucleotides are capable of serving asbarcodes or partial barcodes. In some cases, barcodes or partialbarcodes are obtained from arrays are of known sequence. The use ofknown sequences, including those obtained from an array, for example,may be beneficial in avoiding sequencing errors associated with barcodesof unknown sequence. A microarray may provide at least about 10,000,000,at least about 1,000,000, at least about 900,000, at least about800,000, at least about 700,000, at least about 600,000, at least about500,000, at least about 400,000, at least about 300,000, at least about200,000, at least about 100,000, at least about 50,000, at least about10,000, at least about 1,000, at least about 100, or at least about 10different sequences that may be used as barcodes or partial barcodes.

The beads provided herein may be attached to oligonucleotide sequencesthat may behave as unique identifiers (e.g., barcodes). Often, apopulation of beads provided herein contains a diverse library ofbarcodes, wherein each bead is attached to multiple copies of a singlebarcode sequence. In some cases, the barcode sequences arepre-synthesized and/or designed with known sequences. In some cases,each bead within the library is attached to a unique barcode sequence.In some cases, a plurality of beads will have the same barcode sequenceattached to them. For example, in some cases about 1%, 2%, 3%, 4%, 5%,10%, 20%, 25%, 30%, 50%, 75%, 80%, 90%, 95%, or 100% of the beads in alibrary are attached to a barcode sequence that is identical to abarcode sequence attached to a different bead in the library. Sometimes,about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, or 30% of the beads areattached to the same barcode sequence.

The length of a barcode sequence may be any suitable length, dependingon the application. In some cases, a barcode sequence may be about 2 toabout 500 nucleotides in length, about 2 to about 100 nucleotides inlength, about 2 to about 50 nucleotides in length, about 2 to about 20nucleotides in length, about 6 to about 20 nucleotides in length, orabout 4 to 16 nucleotides in length. In some cases, a barcode sequenceis about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100, 150, 200,250, 300, 400, or 500 nucleotides in length. In some cases, a barcodesequence is greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85,90, 95, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 5000, or 10000nucleotides in length. In some cases, a barcode sequence is less thanabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100, 150, 200,250, 300, 400, 500, 750, or 1000 nucleotides in length.

The barcodes may be loaded into beads so that one or more barcodes areintroduced into a particular bead. In some cases, each bead may containthe same set of barcodes. In other cases, each bead may containdifferent sets of barcodes. In other cases, each bead may comprise a setof identical barcodes. In other cases, each bead may comprise a set ofdifferent barcodes.

The beads provided herein may be attached to oligonucleotide sequencesthat are random, pseudo-random, or targeted N-mers capable of priming asample (e.g., genomic sample) in a downstream process. In some cases,the same n-mer sequences will be present on the oligonucleotidesattached to a single bead or bead population. This may be the case fortargeted priming methods, e.g., where primers are selected to targetcertain sequence segments within a larger target sequence. In othercases, each bead within a population of beads herein is attached to alarge and diverse number of N-mer sequences to, among other things,diversify the sampling of these primers against template molecules, assuch random n-mer sequences will randomly prime against differentportions of the sample nucleic acids.

The length of an N-mer may vary. In some cases, an N-mer (e.g., a randomN-mer, a pseudo-random N-mer, or a targeted N-mer) may be between about2 and about 100 nucleotides in length, between about 2 and about 50nucleotides in length, between about 2 and about 20 nucleotides inlength, between about 5 and about 25 nucleotides in length, or betweenabout 5 and about 15 nucleotides in length. In some cases, an N-mer(e.g., a random N-mer, a pseudo-random N-mer, or a targeted N-mer) maybe about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100, 150, 200,250, 300, 400, or 500 nucleotides in length. In some cases, an N-mer(e.g., a random N-mer, a pseudo-random N-mer, or targeted a N-mer) maybe greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95,100, 150, 200, 250, 300, 400, 500, 750, 1000, 5000, or 10000 nucleotidesin length. In some cases, an N-mer (e.g., a random N-mer, apseudo-random N-mer, or a targeted N-mer) may be less than about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100, 150, 200, 250, 300,400, 500, 750, or 1000 nucleotides in length.

N-mers (including random N-mers) can be engineered for priming aspecific sample type. For example, N-mers of different lengths may begenerated for different types of sample nucleic acids or differentregions of a sample nucleic acid, such that each N-mer lengthcorresponds to each different type of sample nucleic acid or eachdifferent region of a sample nucleic acid. For example, an N-mer of onelength may be generated for sample nucleic acid originating from thegenome of one species (e.g., for example, a human genome) and an N-merof another length may be generated for a sample nucleic acid originatingfrom another species (e.g., for example, a yeast genome). In anotherexample, an N-mer of one length may be generated for sample nucleic acidcomprising a particular sequence region of a genome and an N-mer ofanother length may be generated for a sample nucleic acid comprisinganother sequence region of the genome. Moreover, in addition or as analternative to N-mer length, the base composition of the N-mer (e.g., GCcontent of the N-mer) may also be engineered to correspond to aparticular type or region of a sample nucleic acid. Base content mayvary in a particular type of sample nucleic acid or in a particularregion of a sample nucleic acid, for example, and, thus, N-mers ofdifferent base content may be useful for priming different sample typesof nucleic acid or different regions of a sample nucleic acid.

Populations of beads described elsewhere herein can be generated with anN-mer engineered for a particular sample type or particular samplesequence region. In some cases, a mixed population of beads (e.g., amixture of beads comprising an N-mer engineered for one sample type orsequence region and beads comprising another N-mer engineered foranother sample type or sequence region) with respect to N-mer length andcontent may be generated. In some cases, a population of beads may begenerated, where one or more of the beads can comprise a mixedpopulation of N-mers engineered for a plurality of sample types orsequence regions.

As noted previously, in some cases, the N-mers, whether random ortargeted, may comprise nucleotide analogues, mimics, or non-nativenucleotides, in order to provide primers that have improved performancein subsequent processing steps. For example, in some cases, it may bedesirable to provide N-mer primers that have different melting/annealingprofiles when subjected to thermal cycling, e.g., during amplification,in order to enhance the relative priming efficiency of the n-mersequence. In some cases, nucleotide analogues or non-native nucleotidesmay be incorporated into the N-mer primer sequences in order to alterthe melting temperature profile of the primer sequence as compared to acorresponding primer that includes native nucleotides. In certain cases,the primer sequences, such as the N-mer sequences described herein, mayinclude modified nucleotides or nucleotide analogues, e.g., LNA bases,at one or more positions within the sequence, in order to provideelevated temperature stability for the primers when hybridized to atemplate sequence, as well as provide generally enhanced duplexstability. In some cases, LNA nucleotides are used in place of the A orT bases in primer synthesis to replace those weaker binding bases withtighter binding LNA analogues. By providing enhanced hybridizing primersequences, one may generate higher efficiency amplification processesusing such primers, as well as be able to operate within differenttemperature regimes.

Other modifications may also be provided to the oligonucleotidesdescribed above. For example, in some cases, the oligonucleotides may beprovided with protected termini or other regions, in order to prevent orreduce any degradation of the oligonucleotides, e.g., through anypresent exonuclease activity. In one example, the oligonucleotides maybe provided with one or more phosphorothioate nucleotide analogue at oneor more positions within the oligonucleotide sequence, e.g., adjacent orproximal to the 3′ and/or 5′ terminal position. These phosphorothioatenucleotides typically provide a sulfur group in place of the non-linkingoxygen in an internucleotide linkage within the oligonucleotide toreduce or eliminate nuclease activity on the oligonucleotides,including, e.g., 3′-5′ and/or 5′-3′ exonucleases. In general,phosphorothioate analogues are useful in imparting exo and/orendonuclease resistance to oligonucleotides that include them, includingproviding protection against, e.g., 3′-5′ and/or 5′-3′ exonucleasedigestion of the oligonucleotides. Accordingly, in some aspects, theseone or more phosphorothioate linkages will be in one or more of the last5 to 10 internucleotide linkages at either the 3′ or the 5′ terminus ofthe oligonucleotides, and preferably include one or more of the last 3′or 5′ terminal internucleotide linkage and second to last 5′ terminalinternucleotide linkage, in order to provide protection against 3′-5′ or5′-3′ exonuclease activity. Other positions within the oligonucleotidesmay also be provided with phosphorothiate linkages as well. In additionto providing such protection on the oligonucleotides that comprise thebarcode sequences (and any associated functional sequences), the abovedescribed modifications are also useful in the context of the blockersequences described herein, e.g., incorporating phosphorothioateanalogues within the blocker sequences, e.g., adjacent or proximal tothe 3′ and/or 5′ terminal position as well as potentially otherpositions within the oligonucleotides.

Attaching Content to Pre-Functionalized Beads

A variety of content may be attached to the beads described herein,including beads functionalized with oligonucleotides. Often,oligonucleotides are attached, particularly oligonucleotides withdesired sequences (e.g., barcodes, random N-mers). In many of themethods provided herein, the oligonucleotides are attached to the beadsthrough a primer extension reaction. Beads pre-functionalized withprimer can be contacted with oligonucleotide template. Amplificationreactions may then be performed so that the primer is extended such thata copy of the complement of the oligonucleotide template is attached tothe primer. Other methods of attachment are also possible such asligation reactions.

In some cases, oligonucleotides with different sequences (or the samesequences) are attached to the beads in separate steps. For example, insome cases, barcodes with unique sequences are attached to beads suchthat each bead has multiple copies of a first barcode sequence on it. Ina second step, the beads can be further functionalized with a secondsequence. The combination of first and second sequences may serve as aunique barcode, or unique identifier, attached to a bead. The processmay be continued to add additional sequences that behave as barcodesequences (in some cases, greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10barcode sequences are sequentially added to each bead). The beads mayalso be further functionalized random N-mers that can, for example, actas a random primer for downstream whole genome amplification reactions.

In some cases, after functionalization with a certain oligonucleotidesequence (e.g., barcode sequence), the beads may be pooled and thencontacted with a large population of random Nmers that are then attachedto the beads. In some cases, particularly when the beads are pooledprior to the attachment of the random Nmers, each bead has one barcodesequence attached to it, (often as multiple copies), but many differentrandom Nmer sequences attached to it. FIGS. 4A-4N provide a step-by-stepdepiction of one example method, an example limiting dilution method,for attaching oligonucleotides, such as barcodes and Nmers, to beads.

Limiting dilution may be used to attach oligonucleotides to beads, suchthat the beads, on average, are attached to no more than one uniqueoligonucleotide sequence such as a barcode. Often, the beads in thisprocess are already functionalized with a certain oligonucleotide, suchas primers. For example, beads functionalized with primers (e.g., suchas universal primers) and a plurality of template oligonucleotides maybe combined, often at a high ratio of beads: template oligonucleotides,to generate a mixture of beads and template oligonucleotides. Themixture may then be partitioned into a plurality of partitions (e.g.,aqueous droplets within a water-in-oil emulsion), such as by a bulkemulsification process, emulsions within plates, or by a microfluidicdevice, such as, for example, a microfluidic droplet generator. In somecases, the mixture can be partitioned into a plurality of partitionssuch that, on average, each partition comprises no more than onetemplate oligonucleotide.

Moreover, the template oligonucleotides can be amplified (e.g., viaprimer extension reactions) within the partitions via the primersattached to the beads. Amplification can result in the generation ofbeads comprising amplified template oligonucleotides. Followingamplification, the contents of the partitions may be pooled into acommon vessel (e.g., a tube, a well, etc.). The beads comprising theamplified template oligonucleotides may then be separated from the othercontents of the partitions (including beads that do not compriseamplified template oligonucleotides) by any suitable method including,for example, centrifugation and magnetic separation, with or without theaid of a capture moiety as described elsewhere herein.

Beads comprising amplified template oligonucleotides may be combinedwith additional template oligonucleotides to generate a bulk mixturecomprising the beads and the additional template oligonucleotides. Theadditional template oligonucleotides may comprise a sequence that is atleast partially complementary to the amplified template oligonucleotideson the beads, such that the additional template oligonucleotidehybridizes to the amplified template oligonucleotides. The amplifiedtemplate oligonucleotides can then be extended via the hybridizedadditional template oligonucleotides in an amplification reaction, suchthat the complements of the additional template oligonucleotides areattached to the amplified template oligonucleotides. The cycle ofbinding additional template oligonucleotides to amplifiedoligonucleotides, followed by extension of the amplifiedoligonucleotides in an amplification reaction, can be repeated for anydesired number of additional oligonucleotides that are to be added tothe bead.

The oligonucleotides attached to the amplified template oligonucleotidesmay comprise, for example, one or more of a random N-mer sequence, apseudo random N-mer sequence, or a primer binding site (e.g., auniversal sequence portion, such as a universal sequence portion that iscompatible with a sequencing device). Any of these sequences or anyother sequence attached to a bead may comprise at least a subsection ofuracil containing nucleotides, as described elsewhere herein.

An example of a limiting dilution method for attaching a barcodesequence and a random N-mer to beads is shown in FIGS. 4A-4N. As shownin FIG. 4A, beads 401, (e.g., disulfide cross-linked polyacrylamide gelbeads) are pre-functionalized with a first primer 403. The first primer403 may be, for example, coupled to the beads via a disulfide linkage402 with an acrydite moiety bound to the surface of the beads 401. Insome cases, though, first primer 403 may be coupled to a bead via anacrydite moiety, without a disulfide linkage 402. The first primer 403may be a universal primer for priming template sequences ofoligonucleotides to be attached to the beads and/or may be a primerbinding site (e.g., P5) for use in sequencing an oligonucleotide thatcomprises first primer 403.

The first primer 403 functionalized beads 401 can then be mixed in anaqueous solution with template oligonucleotides (e.g., oligonucleotidescomprising a first primer binding site 404 (e.g., P5c), a templatebarcode sequence 405, and a template primer binding site 407 (e.g.,R1c)) and reagents necessary for nucleic acid amplification (e.g.,dNTPs, polymerase, co-factors, etc.) as shown in FIG. 4B. The aqueousmixture may also comprise a capture primer 406 (e.g., sometimes referredto as a read primer) linked to a capture moiety (e.g., biotin),identical in sequence to the template primer binding site 407 of thetemplate oligonucleotide.

The aqueous mixture is then emulsified in a water/oil emulsion togenerate aqueous droplets (e.g., the droplets comprising one or morebeads 401, a template oligonucleotide, reagents necessary for nucleicamplification, and, if desired, any capture primers 406) in a continuousoil phase. In general, the droplets comprise, on average, at most onetemplate oligonucleotide per droplet. As shown in FIGS. 4B and 4C, afirst round of thermocycling of the droplets results in priming of thetemplate oligonucleotides at primer binding site 404 by first primer 403and extension of first primer 403 such that oligonucleotidescomplementary to the template oligonucleotide sequences are attached tothe gel beads at first primer 403. The complementary oligonucleotidescomprises first primer 403, a barcode sequence 408 (e.g., complementaryto template barcode sequence 405), and a capture primer binding site 415complementary to both template primer binding site 407 and captureprimer 406. Capture primer binding site 415 may also be used as a readprimer binding site (e.g., R1) during sequencing of the complementaryoligonucleotide.

As shown in FIG. 4D, capture primer 406 can bind to capture primerbinding site 415 during the next round of thermocycling. Capture primer406, comprising a capture moiety (e.g., biotin) at its 5′ end, can thenbe extended to generate additional template oligonucleotides (e.g.,comprising sequences 404, 405, and 406), as shown in FIG. 4E.Thermocyling may continue for a desired number of cycles (e.g., at leastabout 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more cycles) up untilall first primer 403 sites of beads 401 are linked to a barcode sequence408 and a capture primer binding site 415. Because each dropletgenerally comprises one or zero template oligonucleotides to start, eachdroplet will generally comprise beads attached to multiple copies of asequence complementary to the template oligonucleotide or no copies of asequence complementary to the template oligonucleotide. At theconclusion of thermocycling, the oligonucleotide products attached tothe beads are hybridized to template oligonucleotides also comprisingthe capture moiety (e.g., biotin), as shown in FIG. 4E.

The emulsion may then be broken via any suitable means and the releasedbeads can be pooled into a common vessel. Using a capture bead (or otherdevice, including capture devices described herein) 409 linked to amoiety (e.g., streptavidin) capable of binding with the capture moietyof capture primer 406, positive beads (e.g., beads comprising sequences403, 408, and 415) may be enriched from negative beads (e.g., beads notcomprising sequences 403, 408, and 415) by interaction of the capturebead with the capture moiety, as shown in FIGS. 4F and 4G. In caseswhere capture beads are used, the beads may be magnetic, such that amagnet may be used for enrichment. As an alternative, centrifugation maybe used for enrichment. Upon enrichment of the positive beads, thehybridized template oligonucleotides comprising the capture moiety andlinked to the capture bead may be denatured from the bead-boundoligonucleotide via heat or chemical means, including chemical meansdescribed herein, as shown in FIG. 4H. Denatured oligonucleotides (e.g.,oligonucleotides comprising sequences 404, 405 and 406) may then beseparated from the positive beads via the capture beads attached to thedenatured oligonucleotides. As shown in FIG. 4H, beads comprisingsequences 403, 408, and 415 are obtained. As an alternative to capturebeads, positive beads may also be sorted from positive beads via flowcytometry by including, for example, an optically active dye inpartitions capable of binding to beads or species coupled to beads.

In bulk aqueous fluid, the beads comprising sequences 403, 408, and 415can then be combined with template random sequences (e.g., randomN-mers) 413 each linked to a sequence 412 complementary to captureprimer binding site 415, as shown in FIG. 4I. As shown in FIG. 4J,capture primer binding site 415 can prime oligonucleotides comprisingtemplate random sequences 413 at sequence 412 upon heating. Followingpriming, capture primer binding site 415 can be extended (e.g., viapolymerase) to link capture primer binding site 415 with a randomsequence 414 that is complementary to template random sequence 413.Oligonucleotides comprising template random sequences 413 and sequence412 can be denatured from the bead using heat or chemical means,including chemical means described herein. Centrifugation and washing ofthe beads, for example, may be used to separate the beads from denaturedoligonucleotides. Following removal of the denatured oligonucleotides,beads comprising a barcode sequence 408 and a random sequence 414 areobtained, as shown in FIGS. 4K, 4L, and 4M. Because the attachment ofrandom sequence 414 was done in bulk, each bead that comprises multiplecopies of a unique barcode sequence 408, also comprises various randomsequences 414.

To release bead-bound oligonucleotides from the beads, stimuli describedelsewhere herein, such as, for example, a reducing agent, may be used.As shown in FIG. 4N, contact of a bead comprising disulfide bonds andlinkages to oligonucleotides via disulfide bonds with a reducing agentdegrades both the bead and the disulfide linkages freeing theoligonucleotide from the bead. Contact with a reducing agent may becompleted, for example, in another partition (e.g., a droplet of anotheremulsion), such that, upon oligonucleotide release from the bead, eachdroplet generally comprises free oligonucleotides all comprising thesame barcode sequence 408, yet various random sequences 414. Via randomsequence 414 acting as a random primer, free oligonucleotides may beused to barcode different regions of a sample nucleic acid also in thepartition. Amplification or ligation schemes, including those describedherein, may be used to complete attachment of barcodes to the samplenucleic acid.

With limiting dilution, the partitions (e.g., droplets) may contain onaverage at most one oligonucleotide sequence per partition. Thisfrequency of distribution at a given sequence-bead dilution followsPoisson distribution. Thus, in some cases, about 6%, 10%, 18%, 20%, 30%,36%, 40%, or 50% of the droplets or partitions may comprise one or feweroligonucleotide sequences. In some cases, more than about 6%, 10%, 18%,20%, 30%, 36%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,or more of the droplets may comprise one or fewer oligonucleotidesequences. In other cases, less than about 6%, 10%, 18%, 20%, 30%, 36%,40%, or 50% of the droplets may comprise one or fewer oligonucleotidesequences.

In some cases, limiting dilution steps may be repeated, prior to theaddition of a random N-mer sequence in order to increase the number ofpositive beads with copies of barcodes. For example, a limiting dilutioncould be prepared such that a desired fraction (e.g., 1/10 to ⅓) ofemulsion droplets comprises a template for amplification. Positive beadscould be generated via amplification of the template (as depicted inFIGS. 4A-4N) such that positives generally comprise no more primer foramplification (e.g., all P5 primer sites have been extended). Theemulsion droplets can then be broken, and subsequently re-emulsifiedwith fresh template at limiting dilution for a second round ofamplification. Positive beads generated in the first round ofamplification generally would not participate in further amplificationbecause their priming sites would already be occupied. The process ofamplification followed by re-emulsification can be repeated for asuitable number of steps, until the desired fraction of positive beadsis obtained.

In some cases, negative beads obtained during sorting after a limitingdilution functionalization may be recovered and further processed togenerate additional positive beads. For example, negative beads may bedispensed into wells of a plate (e.g., a 384 well plate) after recoverysuch that each well generally comprises 1 bead. In some cases,dispensing may be achieved with the aid of flow cytometry (e.g., a flowcytometer directs each negative bead into a well during sorting—anexample flow cytometer being a BD FACS Jazz) or via a dispensing device,such as for example, a robotic dispensing device. Each well can alsocomprise a template barcode sequence and the process depicted in FIGS.4A-4N repeated, except that each well partitions each bead, rather thana fluidic droplet. Because each well comprises template and a bead, eachwell can produce a positive bead. The beads can then be pooled from eachwell and additional sequences (e.g., a random N-mer sequence) can beadded in bulk as described elsewhere herein.

The barcodes may be loaded into the beads at an expected or predictedratio of barcodes per bead to be barcoded. In some cases, the barcodesare loaded such that a ratio of about 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 20000, 50000,100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000,500000000, 1000000000, 5000000000, 10000000000, 50000000000, or100000000000 barcodes are loaded per bead. In some cases, the barcodesare loaded such that a ratio of more than 0.0001, 0.001, 0.1, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 20000, 50000,100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000,1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000,70000000, 80000000, 90000000, 100000000, 200000000, 300000000,400000000, 500000000, 600000000, 700000000, 800000000, 900000000,1000000000, 2000000000, 3000000000, 4000000000, 5000000000, 6000000000,7000000000, 8000000000, 9000000000, 10000000000, 20000000000,30000000000, 40000000000, 50000000000, 60000000000, 70000000000,80000000000, 90000000000, 100000000000 or more barcodes are loaded perbead. In some cases, the barcodes are loaded such that a ratio of lessthan about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007,0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,0.009, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000,500000, 1000000, 5000000, 10000000, 50000000, 100000000, 500000000,1000000000, 5000000000, 10000000000, 50000000000, or 100000000000barcodes are loaded per bead.

Beads, including those described herein (e.g., substantially dissolvablebeads, in some cases, substantially dissolvable by a reducing agent),may be covalently or non-covalently linked to a plurality ofoligonucleotides, wherein at least a subset of the oligonucleotidescomprises a constant region or domain (e.g., a barcode sequence, abarcode domain, a common barcode domain, or other sequence that isconstant among the oligonucleotides of the subset) and a variable regionor domain (e.g., a random sequence, a random N-mer, or other sequencethat is variable among the oligonucleotides of the subset). In somecases, the oligonucleotides may be releasably coupled to a bead, asdescribed elsewhere herein. Oligonucleotides may be covalently ornon-covalently linked to a bead via any suitable linkage, includingtypes of covalent and non-covalent linkages described elsewhere herein.In some cases, an oligonucleotide may be covalently linked to a bead viaa cleavable linkage such as, for example, a chemically cleavable linkage(e.g., a disulfide linkage), a photocleavable linkage, or a thermallycleavable linkage. Beads may comprise more than about or at least about1, 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000,5000000, 10000000, 50000000, 100000000, 500000000, 1000000000,5000000000, 10000000000, 50000000000, 100000000000, 500000000000, or1000000000000 oligonucleotides comprising a constant region or domainand a variable region or domain.

In some cases, the oligonucleotides may each comprise an identicalconstant region or domain (e.g., an identical barcode sequence,identical barcode domain, a common domain, etc.). In some cases, theoligonucleotides may each comprise a variable domain with a differentsequence. In some cases, the percentage of the oligonucleotides thatcomprise an identical constant region (or common domain) may be at leastabout 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some cases, thepercentage of the oligonucleotides that comprise a variable region witha different sequence may be at least about 0.01%, 0.1%, 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. In some cases, the percentage of beads in aplurality of beads that comprise oligonucleotides with differentnucleotide sequences (including those comprising a variable and constantregion or domain) is at least about 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%. In some cases, the oligonucleotides may also comprise oneor more additional sequences, such as, for example a primer binding site(e.g., a sequencing primer binding site), a universal primer sequence(e.g., a primer sequence that would be expected to hybridize to andprime one or more loci on any nucleic acid fragment of a particularlength, based upon the probability of such loci being present within asequence of such length) or any other desired sequence including typesof additional sequences described elsewhere herein.

As described elsewhere herein, a plurality of beads may be generated toform, for example, a bead library (e.g., a barcoded bead library). Insome cases, the sequence of a common domain (e.g., a common barcodedomain) or region may vary between at least a subset of individual beadsof the plurality. For example, the sequence of a common domain or regionbetween individual beads of a plurality of beads may be differentbetween 2 or more, 10 or more, 50 or more, 100 or more, 500 or more,1000 or more, 5000 or more, 10000 or more, 50000 or more, 100000 ormore, 500000 or more, 1000000 or more, 5000000 or more, 10000000 ormore, 50000000 or more, 100000000 or more, 500000000 or more, 1000000000or more, 5000000000 or more, 10000000000 or more, 50000000000 or more,or 100000000000 or more beads of the plurality. In some cases, each beadof a plurality of beads may comprise a different common domain orregion. In some cases, the percentage of individual beads of a pluralityof beads that comprise a different common domain or region may be atleast about 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In somecases, a plurality of beads may comprise at least about 2, 10, 50, 100,500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000,10000000, 50000000, 100000000, 500000000, or more different commondomains coupled to different beads in the plurality.

As an alternative to limiting dilution (e.g., via droplets of anemulsion), other partitioning methods may be used to attacholigonucleotides to beads. As shown in FIG. 13A, the wells of a platemay be used. Beads comprising a primer (e.g., P5, primer linked to thebead via acrydite and, optionally, a disulfide bond) may be combinedwith a template oligonucleotide (e.g., a template oligonucleotidecomprising a barcode sequence) and amplification reagents in the wellsof a plate. Each well can comprise one or more copies of a uniquetemplate barcode sequence and one or more beads. Thermal cycling of theplate extends the primer, via hybridization of the templateoligonucleotide to the primer, such that the bead comprises anoligonucleotide with a sequence complementary to the oligonucleotidetemplate. Thermal cycling may continue for a desired number of cycles(e.g., at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore cycles) up until all primers have been extended.

Upon completion of thermal cycling, the beads may be pooled into acommon vessel, washed (e.g., via centrifugation, magnetic separation,etc.), complementary strands denatured, washed again, and then subjectto additional rounds of bulk processing if desired. For example, arandom N-mer sequence may be added to the bead-bound oligonucleotidesusing the primer extension method described above for limiting dilutionand as shown in FIG. 13B and FIGS. 4I-4M.

As another alternative approach to limiting dilution, a combinatorialprocess involving partitioning in multiwell plates can be used togenerate beads with oligonucleotide sequences as shown in FIG. 13C. Insuch methods, the wells may contain pre-synthesized oligonucleotidessuch as oligonucleotide templates. The beads (e.g., beads withpreincorporated oligonucleotides such as primers) may be divided intothe individual wells of the multiwell plate. For example, a mixture ofbeads containing P5 oligonucleotides may be divided into individualwells of a multiwell plate (e.g., 384 wells), wherein each well containsa unique oligonucleotide template (e.g., an oligonucleotide including afirst partial barcode template or barcode template). A primer extensionreaction may be performed within the individual wells using, forexample, the oligonucleotides templates as the template and the primerattached to the beads as primers. Subsequently, all wells may be pooledtogether and the unreacted products may be removed.

The mixture of beads attached to the amplified product may be re-dividedinto wells of a second multiwell plate (e.g., 384-well plate), whereineach well of the second multiwell plate contains another oligonucleotidesequence (e.g., including a second partial barcode sequence and/or arandom N-mer). In some cases, the oligonucleotide sequence may beattached (e.g., via hybridization) to a blocker oligonucleotide. Withinthe wells of the second multiwell plate, a reaction such as asingle-stranded ligation reaction may be performed to add additionalsequences to each bead (e.g., via ligation of the primer extensionproducts attached to the beads as in the first step with theoligonucleotide in the wells of the second step). In some cases, apartial barcode sequence linked to the bead in the first step is ligatedto a second partial barcode sequence in the second step, to generatebeads comprising full barcode sequences. In some cases, the beadscomprising full barcode sequences also comprise random sequences (e.g.,random N-mers) and/or blocking oligonucleotides. In some cases, a PCRreaction or primer extension reaction is performed to attach theadditional sequence to the beads. Beads from the wells may be pooledtogether, and the unreacted products may be removed. In some cases, theprocess is repeated with additional multi-well plates. The process maybe repeated over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 500,1000, 5000, or 10000 times.

In some combinatorial approaches, ligation methods may be used toassemble oligonucleotide sequences comprising barcode sequences on beads(e.g., degradable beads as described elsewhere herein). For example,separate populations of beads may be provided to which barcodecontaining oligonucleotides are to be attached. These populations mayinclude anchor components (or linkage) for attaching nucleotides, suchas activatable chemical groups (phosphoramidites, acrydite moieties, orother thermally, optically or chemically activatable groups), cleavablelinkages, previously attached oligonucleotide molecules to which thebarcode containing oligonucleotides may be ligated, hybridized, orotherwise attached, DNA binding proteins, charged groups forelectrostatic attachment, or any of a variety of other attachmentmechanisms.

A first oligonucleotide or oligonucleotide segment that includes a firstbarcode sequence segment, is attached to the separate populations, wheredifferent populations include different barcode sequence segmentsattached thereto. Each bead in each of the separate populations may beattached to at least 2, 10, 50, 100, 500, 1000, 5000, 10000, 50000,100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000,500000000, 1000000000, or more first oligonucleotide molecules oroligonucleotide segment molecules. The first oligonucleotide oroligonucleotide segment may be releasably attached to the separatepopulations. In some cases, the first oligonucleotide or oligonucleotidesegments may be attached directly to respective beads in the separatepopulations or may be indirectly attached (e.g., via an anchor componentcoupled to the beads, as described above) to respective beads in theseparate populations.

In some cases, the first oligonucleotide may be attached to the separatepopulations with the aid of a splint (an example of a splint is shown as2306 in FIG. 23A). A splint, as used herein, generally refers to adouble-stranded nucleic acid, where one strand of the nucleic acidcomprises an oligonucleotide to-be-attached to one or more receivingoligonucleotides and where the other strand of the nucleic acidcomprises an oligonucleotide with a sequence that is in partcomplementary to at least a portion of the oligonucleotideto-be-attached and in part complementary to at least a portion of theone or more receiving oligonucleotides. In some cases, anoligonucleotide may be in part complementary to at least a portion of areceiving oligonucleotide via an overhang sequence as shown in FIG.23A). An overhang sequence can be of any suitable length, as describedelsewhere herein.

For example, a splint may be configured such that it comprises the firstoligonucleotide or oligonucleotide segment hybridized to anoligonucleotide that comprises a sequence that is in part complementaryto at least a portion of the first oligonucleotide or oligonucleotidesegment and a sequence (e.g., an overhang sequence) that is in partcomplementary to at least a portion of an oligonucleotide attached tothe separate populations. The splint can hybridize to theoligonucleotide attached to the separate populations via itscomplementary sequence. Once hybridized, the first oligonucleotide oroligonucleotide segment of the splint can then be attached to theoligonucleotide attached to the separate populations via any suitableattachment mechanism, such as, for example, a ligation reaction.

Following attachment of the first oligonucleotide or oligonucleotidesegment to the separate populations, the separate populations are thenpooled to create a mixed pooled population, which is then separated intoa plurality of separate populations of the mixed, pooled population. Asecond oligonucleotide or segment including a second barcode sequencesegment is then attached to the first oligonucleotides on the beads ineach separate mixed, pooled population, such that different mixed pooledbead populations have a different second barcode sequence segmentattached to it. Each bead in the separate populations of the mixed,pooled population may be attached to at least 2, 10, 50, 100, 500, 1000,5000, 10000, 50000, 100000, 500000, 1000000, 5000000, 10000000,50000000, 100000000, 500000000, 1000000000, or more secondoligonucleotide molecules or oligonucleotide segment molecules.

In some cases, the second oligonucleotide may be attached to the firstoligonucleotide with the aid of a splint. For example, the splint usedto attach the first oligonucleotide or oligonucleotide segment to theseparate populations prior to generating the mixed pooled population mayalso comprise a sequence (e.g., an overhang sequence) that is in partcomplementary to at least a portion of the second oligonucleotide. Thesplint can hybridize to the second oligonucleotide via the complementarysequence. Once hybridized, the second oligonucleotide can then beattached to the first oligonucleotide via any suitable attachmentmechanism, such as, for example, a ligation reaction. The splint strandcomplementary to both the first and second oligonucleotides can then bethen denatured (or removed) with further processing. Alternatively, aseparate splint comprising the second oligonucleotide may be provided toattach the second oligonucleotide to the first oligonucleotide inanalogous fashion as described above for attaching the firstoligonucleotide to an oligonucleotide attached to the separatepopulations with the aid of splint. Also, in some cases, the firstbarcode segment of the first oligonucleotide and second barcode segmentof the second oligonucleotide may be joined via a linking sequence asdescribed elsewhere herein.

The separate populations of the mixed, pooled population can then bepooled and the resulting pooled bead population then includes a diversepopulation of barcode sequences, or barcode library that is representedby the product of the number of different first barcode sequences andthe number of different second barcode sequences. For example, where thefirst and second oligonucleotides include, e.g., all 256 4-mer barcodesequence segments, a complete barcode library may include 65,536 diverse8 base barcode sequences.

The barcode sequence segments may be independently selected from a setof barcode sequence segments or the first and second barcode sequencesegments may each be selected from separate sets of barcode sequencesegments. Moreover, the barcode sequence segments may individually andindependently comprise from 2 to 20 nucleotides in length, preferablyfrom about 4 to about 20 nucleotides in length, more preferably fromabout 4 to about 16 nucleotides in length or from about 4 to about 10nucleotides in length. In some cases, the barcode sequence segments mayindividually and independently comprise at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides inlength. In particular, the barcode sequence segments may comprise2-mers, 3-mers, 4-mers, 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers,11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers,19-mers, 20-mers, or longer sequence segments.

Furthermore, the barcode sequence segments included within the first andsecond oligonucleotide sequences or sequence segments will typicallyrepresent at least 10 different barcode sequence segments, at least 50different barcode sequence segments, at least 100 different barcodesequence segments, at least 500 different barcode sequence segments, atleast 1,000 different barcode sequence segments, at least about 2,000different barcode sequence segments, at least about 4,000 differentbarcode sequence segments, at least about 5,000 different barcodesequence segments, at least about 10,000 different barcode sequencesegments, at least 50,000 different barcode sequence segments, at least100,000 barcode sequence segments, at least 500,000 barcode sequencesegments, at least 1,000,000 barcode sequence segments, or more. Inaccordance with the processes described above, these differentoligonucleotides may be allocated amongst a similar or the same numberof separate bead populations in either the first or secondoligonucleotide addition step, e.g., at least 10, 100, 500, 1000, 2000,4000, 5000, 10000, 50000, 100000, 500000, 1000000, etc., differentbarcode sequence segments being separately added to at least 10, 100,500, 1000, 2000, 4000, 5000, 10000, 50000, 100000, 500000, 1000000,etc., separate bead populations.

As a result, resulting barcode libraries may range in diversity of fromat least about 100 different barcode sequence segments to at least about1,000,000, 2,000,000, 5,000,000, 10,000,000 100,000,000 or moredifferent barcode sequence segments as described elsewhere herein, beingrepresented within the library.

As noted previously, either or both of the first and secondoligonucleotide sequences or sequence segments, or subsequently addedoligonucleotides (e.g., addition of a third oligonucleotide to thesecond oligonucleotide, addition of a fourth oligonucleotide to an addedthird oligonucleotide, etc.), may include additional sequences, e.g.,complete or partial functional sequences (e.g., a primer sequence (e.g.,a universal primer sequence, a targeted primer sequence, a random primersequence), a primer annealing sequence, an attachment sequence, asequencing primer sequence, a random N-mer, etc.), for use in subsequentprocessing. These sequences will, in many cases, be common among beadsin the separate populations, subsets of populations, and/or common amongall beads in the overall population. In some cases, the functionalsequences may be variable as between different bead subpopulations,different beads, or even different molecules attached to a single bead.Moreover, either or both of the first and second oligonucleotidesequences or sequence segments may comprise a sequence segment thatincludes one or more of a uracil containing nucleotide and a non-nativenucleotide, as described elsewhere herein. In addition, althoughdescribed as oligonucleotides comprising barcode sequences, it will beappreciated that such references includes oligonucleotides that arecomprised of two, three or more discrete barcode sequence segments thatare separated by one or more bases within the oligonucleotide, e.g., afirst barcode segment separated from a second barcode segment by 1, 2,3, 4, 5, 6, or 10 or more bases in the oligonucleotide in which they arecontained. Preferably, barcode sequence segments will be locatedadjacent to each other or within 6 bases, 4 bases, 3 bases or two basesof each other in the oligonucleotide sequence in which they arecontained. Together, whether contiguous within an oligonucleotidesequence, or separated by one or more bases, such collective barcodesequence segments within a given oligonucleotide are referred to hereinas a barcode sequence, barcode sequence segment, or barcode domain.

An example combinatorial method for generating beads with sequencescomprising barcode sequences as well as specific types of functionalsequences is shown in FIGS. 23A-23D. Although described in terms ofcertain specific sequence segments for purposes of illustration, it willbe appreciated that a variety of different configurations may beincorporated into the barcode containing oligonucleotides attached tothe beads described herein, including a variety of different functionalsequence types, primer types, e.g., specific for different sequencingsystems, and the like. As shown in FIG. 23A, beads 2301 may be generatedand covalently linked (e.g., via an acrydite moiety or other species) toa first oligonucleotide component to be used as an anchoring componentand/or functional sequence or partial functional sequence, e.g., partialP5 sequence 2302. In each well of a plate (e.g., a 384-well plate) anoligonucleotide 2303, comprising the remaining P5 sequence and a uniquefirst partial barcode sequence (indicated by bases “DDDDDD” inoligonucleotide 2303), can be hybridized to an oligonucleotide 2304 thatcomprises the complement of oligonucleotide 2303 and additional basesthat overhang each end of oligonucleotide 2303. Hybridized product (a“splint”) 2306 can thus be generated. Each overhang of the splint can beblocked (indicated with an “X” in FIG. 23A) with a blocking moiety toprevent side product formation. Non-limiting examples of blockingmoieties include 3′ Inverted dT, dideoxycytidine (ddC), and 3′C3 Spacer.Accordingly, in the example described, different splints can begenerated, each with a unique first partial barcode sequence or itscomplement, e.g., 384 different splints, as described.

As shown in FIG. 23B, beads 2301 can be added to each well of the plateand the splint 2306 in each well can hybridize with the correspondinganchor sequence, e.g., partial P5 sequence 2302, of beads 2301, via oneof the overhangs of oligonucleotide 2304. Limited stability of theoverhang of oligonucleotide 2304 in hybridizing partial P5 sequence 2302can permit dynamic sampling of splint 2306, which can aid in ensuringthat subsequent ligation of oligonucleotide 2303 to partial P5 sequence2302 is efficient. A ligation enzyme (e.g., a ligase) can ligate partialP5 sequence 2302 to oligonucleotide 2303. An example of a ligase wouldbe T4 DNA ligase. Following ligation, the products can be pooled and thebeads washed to remove unligated oligonucleotides.

As shown in FIG. 23C, the washed products can then be redistributed intowells of another plate (e.g., a 384-well plate), with each well of theplate comprising an oligonucleotide 2305 that has a unique secondpartial barcode sequence (indicated by “DDDDDD” in oligonucleotide 2305)and an adjacent short sequence (e.g., “CC” adjacent to the secondpartial barcode sequence and at the terminus of oligonucleotide 2305)complementary to the remaining overhang of oligonucleotide 2304.Oligonucleotide 2305 can also comprise additional sequences, such as R1sequences and a random N-mer (indicated by “NNNNNNNNNN” inoligonucleotide 2305). In some cases, oligonucleotide 2305 may comprisea uracil containing nucleotide. In some cases, any of the thyminecontaining nucleotides of oligonucleotide 2305 may be substituted withuracil containing nucleotides. In some cases, in order to improve theefficiency of ligation of the oligonucleotide comprising the secondpartial barcode sequence, e.g., sequence 2305, to the first partialbarcode sequence, e.g., sequence 2303, a duplex strand, e.g., that iscomplementary to all or a portion of oligonucleotide 2305, may beprovided hybridized to some portion or all of oligonucleotide 2305,while leaving the overhang bases available for hybridization to splint2304. As noted previously, splint 2304 and/or the duplex strand, may beprovided blocked at one or both of their 3′ and 5′ ends to preventformation of side products from or between one or both of the splint andthe duplex strand. In preferred aspects, the duplex strand may becomplementary to all or a portion of oligonucleotide 2305. For example,where oligonucleotide 2305 includes a random n-mer, the duplex strandmay be provided that does not hybridize to that portion of theoligonucleotide.

Via the adjacent short sequence, oligonucleotide 2305 can be hybridizedwith oligonucleotide 2304, as shown in FIG. 23C. Again, the limitedstability of the overhang in hybridizing the short complementarysequence of oligonucleotide 2305 can permit dynamic sampling ofoligonucleotide 2305, which can aid in ensuring that subsequent ligationof oligonucleotide 2305 to oligonucleotide 2303 is efficient. A ligationenzyme (e.g., a ligase) can then ligate oligonucleotide 2305 tooligonucleotide 2303. Ligation of oligonucleotide 2305 tooligonucleotide 2303 can result in the generation of a full barcodesequence, via the joining of the first partial barcode sequence ofoligonucleotide 2305 and the second partial barcode sequence ofoligonucleotide 2303. As shown in FIG. 23D, the products can then bepooled, the oligonucleotide 2304 can be denatured from the products, andunbound oligonucleotides can then be washed away. Following washing, adiverse library of barcoded beads can be obtained, with each bead boundto, for example, an oligonucleotide comprising a P5 sequence, a fullbarcode sequence, an R1 sequence, and a random N-mer. In this example,147,456 unique barcode sequences can be obtained (e.g., 384 unique firstpartial barcode sequence×384 unique second partial barcode sequences).

In some cases, the inclusion of overhang bases that aid in ligation ofoligonucleotides as described above can result in products that all havethe same base at a given position, including in between portions of abarcode sequence as shown in FIG. 24A. Limited or no base diversity at agiven sequence position across sequencing reads may result in failedsequencing runs, depending upon the particular sequencing methodutilized. Accordingly, in a number of aspects, the overhang bases may beprovided with some variability as between different splints, either interms of base identity or position within the overall sequenced portionof the oligonucleotide. For example, in a first example, one or morespacer bases 2401 (e.g., “1” “2” in FIG. 24B at 2401) can be added tosome oligonucleotides used to synthesize larger oligonucleotides onbeads, such that oligonucleotide products differ slightly in length fromone another, and thus position the overhang bases at different locationsin different sequences. Complementary spacer bases may also be added tosplints necessary for sequence component ligations. A slight differencein oligonucleotide length between products can result in base diversityat a given read position, as shown in FIG. 24B.

In another example shown in FIGS. 25A-25C, splints comprising a randombase overhang may be used to introduce base diversity at read positionscomplementary to splint overhangs. For example, a double-stranded splint2501 may comprise a random base (e.g., “NN” in FIG. 25A) overhang 2503and a determined base (e.g., “CTCT” in FIG. 25A) overhang 2506 on onestrand and a first partial barcode sequence (e.g., “DDDDDD” in FIG. 25A)on the other strand. Using an analogous ligation scheme as describedabove for the Example depicted in FIGS. 23A-23D, the determined overhang2506 may be used to capture sequence 2502 (which may be attached to abead as shown in FIGS. 23A-23D) via hybridization for subsequentligation with the upper strand (as shown in FIG. 25A) of splint 2501.Although overhang 2506 is illustrated as a four base determined sequenceoverhang, it will be appreciated that this sequence may be longer inorder to improve the efficiency of hybridization and ligation in thefirst ligation step. As such determined base overhang 2506 may include4, 6, 8, 10 or more bases in length that are complementary to partial P5sequence 2502. Moreover, the random base overhang 2503 may be used tocapture the remaining component (e.g., sequence 2504) of the finaldesired sequence. Sequence 2504 may comprise a second partial barcodesequence (“DDDDDD” in sequence 2504 of FIG. 25C), the complement 2505(e.g., “NN” at 2505 in FIG. 25C) of the random base overhang 2503 at oneend and a random N-mer 2507 at its other end (e.g., in sequence 2504 ofFIG. 25C).

Due to the randomness of the bases in random base overhang 2503, basesincorporated into the ligation product at complement 2505 can vary, suchthat products comprise a variety of bases at the read positions ofcomplement 2505. As will be appreciated, in preferred aspects, thesecond partial barcode sequence portion to be ligated to the firstpartial barcode sequence will typically include a population of suchsecond partial barcode sequences that includes all of the complements tothe random overhang sequences, e.g., a given partial barcode sequencewill be present with, e.g., 16 different overhang portions, in order toadd the same second partial barcode sequence to each bead in a givenwell where multiple overhang sequences are represented. While only twobases are shown for random overhang 2503 and complement 2505 in FIGS.25A-25C, the example is not meant to be limiting. Any suitable number ofrandom bases in an overhang may be used. Further, while described asrandom overhang sequences, in some cases, these overhang sequences maybe selected from a subset of overhang sequences. For example, in somecases, the overhangs will be selected from subsets of overhang sequencesthat include fewer than all possible overhang sequences of the length ofthe overhang, which may be more than one overhang sequence, and in somecases, more than 2, more than 4, more than 10, more than 20, more than50, or even more overhang sequences.

In another example, a set of splints, each with a defined overhangselected from a set of overhang sequences of a given length, e.g., a setof at least 2, 4, 10, 20 or more overhang sequences may be used tointroduce base diversity at read positions complementary to splintoverhangs. Again, because these overhangs are used to ligate a secondpartial barcode sequence to the first barcode sequence, it will bedesirable to have all possible overhang complements represented in thepopulation of second partial barcode sequences. As such, in many cases,it will be preferred to keep the numbers of different overhang sequenceslower, e.g., less than 50, less than 20, or in some cases, less than 10or less than 5 different overhang sequences. In many cases, the numberof different linking sequences in a barcode library will be between 2and 4096 different linking sequences, with preferred libraries havingbetween about 2 and about 50 different linking sequences. Likewise itwill typically be desirable to keep these overhang sequences of arelatively short length, in order to avoid introducing non-relevantbases to the ultimate sequence reads. As such, these overhang sequenceswill typically be designed to introduce no more than 10, no more than 9,no more than 8, no more than 7, no more than 6, no more than 5, no morethan 4, and in some cases, 3 or fewer nucleotides to the overalloligonucleotide construct. In some cases, the length of an overhangsequence may be from about 1 to about 10 nucleotides in length, fromabout 2 to about 8 nucleotides in length, from about 2 to about 6nucleotides in length, or from about 2 to about 4 nucleotides in length.In general, each splint in the set can comprise an overhang with adifferent sequence from other splints in the set, such that the base ateach position of the overhang is different from the base in the samebase position in the other splints in the set. An example set of splintsis depicted in FIG. 26. The set comprises splint 2601 (comprising anoverhang of “AC” 2602), splint 2603 (comprising an overhang of “CT”2604), splint 2605 (comprising an overhang of “GA” 2606), and splint2607 (comprising an overhang of “TG” 2608). Each splint can alsocomprise an overhang 2609 (e.g., “CTCT” in each splint) and firstpartial barcode sequence (“DDDDDD”). As shown in FIG. 26, each splintcan comprise a different base in each position of its unique overhang(e.g., overhang 2602 in splint 2601, overhang 2604 in splint 2603,overhang 2606 in splint 2605, and overhang 2608 in splint 2607) suchthat no splint overhang comprises the same base in the same baseposition. Because each splint comprises a different base in eachposition of its unique overhang, products generated from each splint canalso have a different base in each complementary position when comparedto products generated from one of the other splints. Thus, basediversity at these positions can be achieved.

Such products can be generated by hybridizing the first component of thedesired sequence (e.g., sequence 2502 in FIGS. 25B-25C comprising afirst partial barcode sequence; the first component may also be attachedto a bead) with the overhang common to each splint (e.g., overhang 2609in FIG. 26); ligating the first component of the sequence to the splint;hybridizing the second part of the desired sequence (e.g., a sequencesimilar to sequence 2504 in FIG. 25C comprising a second partial barcodesequence, except that the sequence comprises bases complementary to theunique overhang sequence at positions 2505 instead of random bases) tothe unique overhang of the splint; and ligating the second component ofthe desired sequence to the splint. The unligated portion of the splint(e.g., bottom sequence comprising the overhangs as shown in FIG. 26) canthen be denatured, the products washed, etc. as described previously toobtain final products. As will be appreciated, and as noted previously,these overhang sequences may provide 1, 2, 3, 4, 5 or 6 or more basesbetween different partial barcode sequences (or barcode sequencesegments), such that they provide a linking sequence between barcodesequence segments, with the characteristics described above. Such alinking sequence may be of varied length, such as for example, fromabout 2 to about 10 nucleotides in length, from about 2 to about 8nucleotides in length, from about 2 to about 6 nucleotides in length,from about 2 to about 5 nucleotides in length, or from about 2 to about4 nucleotides in length.

An example workflow using the set of splints depicted in FIG. 26 isshown in FIG. 27. For each splint in the set, the splint strandcomprising the unique overhang sequence (e.g., the bottom strand ofsplints shown in FIG. 26) can be provided in each well of one or moreplates. In FIG. 27, two 96-well plates of splint strands comprising aunique overhang sequence are provided for each of the four splint types,for a total of eight plates. Of the eight plates, two plates (2601 a,2601 b) correspond to the bottom strand of splint 2601 comprising aunique overhang sequence (“AC”) in FIG. 26, two plates (2603 a, 2603 b)correspond to the bottom strand of splint 2603 in FIG. 26 comprising aunique overhang sequence (“CT”), two plates (2605 a, 2605 b) correspondto the bottom strand of splint 2605 in FIG. 26 comprising a uniqueoverhang sequence (“GA”), and two plates (2607 a, 2607 b) correspond tothe bottom strand of splint 2607 in FIG. 26 comprising a unique overhangsequence (“TG”). The oligonucleotides in each 96-well plate (2601 a,2601 b, 2603 a, 2603 b, 2605 a, 2605 b, 2607 a, and 2607 b) can betransferred to another set of 96-well plates 2702, with each platetransferred to its own separate plate (again, for a total of eightplates), and each well of each plate transferred to its correspondingwell in the next plate.

The splint strand comprising a unique first partial barcode sequence(e.g., the upper strand of splints shown in FIG. 26) and a first partialP5 sequence can be provided in one or more plates. In FIG. 27, suchsplint strands are provided in two 96-well plates 2708 a and 2708 b,with each well of the two plates comprising an oligonucleotide with aunique first partial barcode sequence, for a total of 192 unique firstpartial barcode sequences across the two plates. Each well of plate 2708a can be added to its corresponding well in four of the plates 2702 andeach well of plate 2708 b can be added to its corresponding well in theother four of the plates 2702. Thus, the two splint strands in each wellcan hybridize to generate a complete splint. After splint generation,each well of two of the 96-well plates 2702 in FIG. 27 comprises asplint configured as splint 2601, splint 2603, splint 2605, or splint2607 in FIG. 26 and a unique first partial barcode sequence, for a totalof 192 unique first partial barcode sequences.

To each of the wells of the plates 2702, beads 2709 comprising a secondpartial P5 sequence (e.g., similar or equivalent to sequence 2502 inFIGS. 25B-25C) can then be added. The splints in each well can hybridizewith the second partial P5 sequence via the common overhang sequence2609 of each splint. A ligation enzyme (e.g., a ligase) can then ligatethe second partial P5 sequence to the splint strand comprising theremaining first partial P5 sequence and the first partial barcodesequence. First products are, thus, generated comprising beads linked toa sequence comprising a P5 sequence and a first partial barcodesequence, still hybridized with the splint strand comprising theoverhang sequences. Following ligation, first products from the wells ofeach plate can be separately pooled to generate plate pools 2703. Theplate pools 2703 corresponding to each two-plate set (e.g., each setcorresponding to a particular splint configuration) can also beseparately pooled to generate first product pools 2704, such that eachfirst product pool 2704 comprises products generated from splintscomprising only one unique overhang sequence. In FIG. 27, four firstproduct pools 2704 are generated, each corresponding to one of the foursplint types used in the example. The products in each plate pool 2703may be washed to remove unbound oligonucleotides, the products in eachfirst product pool 2704 may be washed to remove unboundoligonucleotides, or washing may occur at both pooling steps. In somecases, plate pooling 2703 may be bypassed with the contents of eachtwo-plate set entered directly into a first product pool 2704.

Next, each first product pool 2704 can be aliquoted into each well oftwo 96-well plates 2705, as depicted in FIG. 27, for a total of eightplates (e.g., two plates per product pool 2704). Separately,oligonucleotides that comprise a unique second partial barcode sequence,a terminal sequence complementary to one of the four unique overhangsequences, and any other sequence to be added (e.g., additionalsequencing primer sites, random N-mers, etc.) can be provided in 96-wellplates 2706. Such oligonucleotides may, for example, comprise a sequencesimilar to sequence 2504 in FIG. 25C, except that the sequence comprisesbases complementary to a unique overhang sequence at position 2505instead of random bases. For example, for splint 2601 shown in FIG. 26,the bases in position 2505 would be “TG”, complementary to the uniqueoverhang 2602 (“AC”) of splint 2601. Of the plates 2706, sets of twoplates can each comprise oligonucleotides comprising sequencescomplementary to one of the four unique overhang sequences, for a totalof eight plates and four plate sets as shown in FIG. 27. Plates 2706 canbe configured such that each well comprises a unique second partialbarcode sequence, for a total of 768 unique second partial barcodesequences across the eight plates.

Each plate of plates 2706 can be paired with a corresponding plate ofplates 2705, based on the appropriate unique overhang sequence of firstproducts entered into the plate of plates 2705, as shown in FIG. 27.Oligonucleotides in each well of the plate from plates 2706 can be addedto its corresponding well in its corresponding plate from plates 2705,such that each well comprises an aliquot of first products from theappropriate first product pool 2704 and oligonucleotides comprising aunique second barcode sequence and any other sequence (e.g., randomN-mers) from plates 2706. In each well of the plates 2705, the uniqueoverhang sequence of each first product can hybridize with anoligonucleotide comprising the second partial barcode sequence, via theoligonucleotide's bases complementary to the unique overhang sequence. Aligation enzyme (e.g., a ligase) can then ligate the oligonucleotides tothe first products. Upon ligation, second products comprising completebarcode sequences are generated via joining of the first partial barcodesequence of the first products with the second partial barcode sequenceof the second products. The second products obtained from plates 2705can be removed and deposited into a common second product pool 2707. Thesplint strands comprising the overhangs (as shown in FIG. 26) can thenbe denatured in product pool 2707, and the products washed to obtainfinal products. A total of 147,456 unique barcode sequences can beobtained (e.g., 192 first partial barcode sequences×768 second partialbarcode sequences) with base diversity in base positions complementaryto unique overhang sequences used during ligations.

The above example with respect to splint sets is not meant to belimiting, nor is the number and type (s) of plates used forcombinatorial synthesis. A set of splints can comprise any suitablenumber of splints. Moreover, each set of splints may be designed withthe appropriate first partial barcode sequence diversity depending upon,for example, the number of unique barcode sequences desired, the numberof bases used to generate a barcode sequence, etc.

Using a combinatorial plate method, libraries of barcoded beads withhigh-diversity can be generated. For example, if two 384-well plates areused, each with oligonucleotides comprising partial barcode sequencespre-deposited in each well, it is possible that 384×384 or 147,456unique barcode sequences can be generated. The combinatorial examplesshown herein are not meant to be limiting as any suitable combination ofplates may be used. For example, while in some cases, the barcodesequence segments added in each combinatorial step may be selected fromthe same sets of barcode sequence segments. However, in many cases, thebarcode sequence segments added in each combinatorial step may beselected from partially or completely different sets of oligonucleotidesequences. For example, in some cases, a first oligonucleotide segmentmay include a barcode sequence from a first set of barcode sequences,e.g., 4-mer sequences, while the second oligonucleotide sequence mayinclude barcode sequences from a partially or completely different setof barcode sequence segments, e.g., 4-mer sequences, 6-mer sequences,8-mer sequences, etc., or even sequences of mixed lengths, e.g., wherethe second oligonucleotide segment is selected form a set ofoligonucleotides having barcode sequences having varied lengths andsequences, to generate multiparameter variability in the generatedbarcodes, e.g., sequence and length.

With reference to the example above, for example, the number and type ofplates (and barcodes) used for each step in a combinatorial method doesnot have to be the same. For example, a 384 well plate may be used for afirst step and a 96 well plate may be used for a second step for a totalof 36,864 unique barcode sequences generated. Furthermore, the number ofbases of a full barcode sequence added in each combinatorial step doesnot need to be the same. For example, in a first combinatorial step, 4bases of a 12 base barcode sequence may be added, with the remaining 8bases added in a second combinatorial step. Moreover, the number ofcombinatorial steps used to generate a full barcode sequence may alsovary. In some cases, about 2, 3, 4, 5, 6, 7, 8, 9, or 10 combinatorialsteps are used.

The primer extension reactions and ligation reactions can be conductedwith standard techniques and reagents in the multiwell plates. Forexample, the polymer, poly-ethylene glycol (PEG), may be present duringthe single-stranded ligation reaction at a concentration of about 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In somecases, the PEG may be present during the ligation reaction at aconcentration of more than about 6%, 10%, 18%, 20%, 30%, 36%, 40%, 50%or more. In some cases, the PEG may be present during the ligationreaction in the second plate at a concentration of less than about 6%,10%, 18%, 20%, 30%, 36%, 40%, or 50%.

The methods provided herein may reduce nucleotide bias in ligationreactions. Better results may occur when the first extension in thefirst well plate may be run to completion. For the single-strandligation step in the second well plate, no competition may be presentwhen only one type of oligonucleotide sequence is used. The partitioningin wells method for attaching content to beads may avoid misformedadaptors with 8N ends, particularly when the first extension in thefirst well plate is run to completion.

Potential modifications to the partitioning in wells process may includereplacing the single-strand ligation step with PCR by providing thesecond oligonucleotide sequence with degenerate bases, modifying thefirst oligonucleotide sequence to be longer than the secondoligonucleotide sequence, and/or adding a random N-mer sequence in aseparate bulk reaction after the single-strand ligation step, as thismay save synthesis costs and may reduce N-mer sequence bias.

In some cases, the following sequence of processes may be used to attacha barcode sequence to a bead. The barcode sequence may be mixed withsuitable PCR reagents and a plurality of beads in aqueous fluid. Theaqueous fluid may be emulsified within an immiscible fluid, such as anoil, to form an emulsion. The emulsion may generate individual fluidicdroplets containing the barcode sequence, the bead, and PCR reagents.Individual fluidic droplets may be exposed to thermocycling conditions,in which the multiple rounds of temperature cycling permits priming andextension of barcode sequences. The emulsion containing the fluidicdroplets may be broken by continuous phase exchange, described elsewherein this disclosure. Resulting barcoded beads suspended in aqueoussolution may be sorted by magnetic separation or other sorting methodsto obtain a collection of purified barcoded beads in aqueous fluid.

In some cases, the following sequence of processes may be used to attachan N-mer sequence to a bead. The N-mer sequence may be mixed withsuitable PCR reagents and a plurality of pooled barcoded beads inaqueous fluid. The aqueous fluid may be heated to permit hybridizationand extension of the N-mer sequence. Additional heating may permitremoval of the complement strand.

The PCR reagents may include any suitable PCR reagents. In some cases,dUTPs may be substituted for dTTPs during the primer extension or otheramplification reactions, such that oligonucleotide products compriseuracil containing nucleotides rather than thymine containingnucleotides. This uracil-containing section of the universal sequencemay later be used together with a polymerase that will not accept orprocess uracil-containing templates to mitigate undesired amplificationproducts.

Amplification reagents may include a universal primer, universal primerbinding site, sequencing primer, sequencing primer binding site,universal read primer, universal read binding site, or other primerscompatible with a sequencing device, e.g., an Illumina sequencer, IonTorrent sequencer, etc. The amplification reagents may include P5, noncleavable 5′acrydite-P5, a cleavable 5′ acrydite-SS-P5, R1c, Biotin R1c,sequencing primer, read primer, P5_Universal, P5_U, 52-BioR1-rc, arandom N-mer sequence, a universal read primer, etc. In some cases, aprimer may contain a modified nucleotide, a locked nucleic acid (LNA),an LNA nucleotide, a uracil containing nucleotide, a nucleotidecontaining a non-native base, a blocker oligonucleotide, a blocked 3′end, 3′ddCTP. FIG. 19 provides additional examples.

As described herein, in some cases oligonucleotides comprising barcodesare partitioned such that each bead is partitioned with, on average,less than one unique oligonucleotide sequence, less than two uniqueoligonucleotide sequences, less than three unique oligonucleotidesequences, less than four unique oligonucleotide sequences, less thanfive unique oligonucleotide sequences, or less than ten uniqueoligonucleotide sequences. Therefore, in some cases, a fraction of thebeads does not contain an oligonucleotide template and therefore cannotcontain an amplified oligonucleotide. Thus, it may be desirable toseparate beads comprising oligonucleotides from beads not comprisingoligonucleotides. In some cases, this may be done using a capturemoiety.

In some embodiments, a capture moiety may be used with isolation methodssuch as magnetic separation to separate beads containing barcodes frombeads, which may not contain barcodes. As such, in some cases, theamplification reagents may include capture moieties attached to a primeror probe. Capture moieties may allow for sorting of labeled beads fromnon-labeled beads to confirm attachment of primers and downstreamamplification products to a bead. Exemplary capture moieties includebiotin, streptavidin, glutathione-S-transferase (GST), cMyc, HA, etc.The capture moieties may be, or include, a fluorescent label or magneticlabel. The capture moiety may comprise multiple molecules of a capturemoiety, e.g., multiple molecules of biotin, streptavidin, etc. In somecases, an amplification reaction may make use of capture primersattached to a capture moiety (as described elsewhere herein), such thatthe primer hybridizes with amplification products and the capture moietyis integrated into additional amplified oligonucleotides duringadditional cycles of the amplification reaction. In other cases, a probecomprising a capture moiety may be hybridized to amplifiedoligonucleotides following the completion of an amplification reactionsuch that the capture moiety is associated with the amplifiedoligonucleotides.

A capture moiety may be a member of binding pair, such that the capturemoiety can be bound with its binding pair during separation. Forexample, beads may be generated that comprise oligonucleotides thatcomprise a capture moiety that is a member of a binding pair (e.g.,biotin). The beads may be mixed with capture beads that comprise theother member of the binding pair (e.g., streptavidin), such that the twobinding pair members bind in the resulting mixture. The bead-capturebead complexes may then be separated from other components of themixture using any suitable means, including, for example centrifugationand magnetic separation (e.g., including cases where the capture bead isa magnetic bead).

In many cases as described, individual beads will generally haveoligonucleotides attached thereto, that have a common overall barcodesequence segment. As described herein, where a bead includesoligonucleotides having a common barcode sequence, it is generally meantthat of the oligonucleotides coupled to a given bead, a significantpercentage, e.g., greater than 70%, greater than 80%, greater than 90%,greater than 95% or even greater than 99% of the oligonucleotides of orgreater than a given length, e.g., including the full expected length orlengths of final oligonucleotides and excluding unreacted anchorsequences or partial barcode sequences, include the same or identicalbarcode sequence segments. This barcode sequence segment or domain(again, which may be comprised of two or more sequence segmentsseparated by one or more bases) may be included among other common orvariable sequences or domains within a single bead. Also as described,the overall population of beads will include beads having large numbersof different barcode sequence segments. In many cases, however, a numberof separate beads within a given bead population may include the samebarcode sequence segment. In particular, a barcode sequence libraryhaving 1000, 10,000, 1,000,000, 10,000,000 or more different sequences,may be represented in bead populations of greater than 100,000,1,000,000, 10,000,000, 100,000,000, 1 billion, 10 billion, 100 billionor more discrete beads, such that the same barcode sequence isrepresented multiple times within a given bead population orsubpopulation. For example, the same barcode sequence may be present ontwo or more beads within a given analysis, 10 or more beads, 100 or morebeads, etc.

A capture device, such as a magnetic bead, with a corresponding linkage,such as streptavidin, may be added to bind the capture moiety, forexample, biotin. The attached magnetic bead may then enable isolation ofthe barcoded beads by, for example, magnetic sorting. Magnetic beads mayalso be coated with other linking entities besides streptavidin,including nickel-IMAC to enable the separation of His-tagged fusionproteins, coated with titanium dioxide to enable the separation ofphosphorylated peptides, or coated with amine-reactive NHS-ester groupsto immobilize protein or other ligands for separation.

In some embodiments, the capture moiety may be attached to a primer, toan internal sequence, to a specific sequence within the amplifiedproduct, to a barcode sequence, to a universal sequence, or to acomplementary sequence. Capture moieties may be attached by PCRamplification or ligation. Capture moieties may include a universal tagsuch as biotin attached to a specific target such as a primer beforeadded to the bead population. In other cases, capture moieties mayinclude a specific tag that recognizes a specific sequence or protein orantibody that may be added to the bead population independently. In someembodiments, the capture moieties may be pre-linked to a sorting bead,such as a magnetic bead. In some cases, the capture moiety may be afluorescent label, which may enable sorting via fluorescence-activatedcell sorting (FACS).

In some cases, a nucleic acid label (e.g., fluorescent label) may beused to identify fluidic droplets, emulsions, or beads that containoligonucleotides. Sorting (e.g., via flow cytometry) of the labeleddroplets or beads may then be performed in order to isolate beadsattached to amplified oligonucleotides. Exemplary stains includeintercalating dyes, minor-groove binders, major groove binders, externalbinders, and bis-intercalators. Specific examples of such dyes includeSYBR green, SYBR blue, DAPI, propidium iodide, SYBR gold, ethidiumbromide, propidium iodide, imidazoles (e.g., Hoechst 33258, Hoechst33342, Hoechst 34580, and DAPI), 7-AAD, SYTOX Blue, SYTOX Green, SYTOXOrange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1,BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1,TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen,OliGreen, RiboGreen, EvaGreen, SYBR Green, SYBR Green II, SYBR DX,SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23,-12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84,-85 (orange), SYTO-64, -17, -59, -61, -62, -60, and -63 (red).

Multi-Functional Beads

Beads may be linked to a variety of species (including non-nucleic acidspecies) such that they are multi-functional. For example, a bead may belinked to multiple types of oligonucleotides comprising a barcodesequence and an N-mer (e.g., a random N-mer or a targeted N-mer asdescribed below). Each type of oligonucleotide may differ in its barcodesequence, its N-mer, or any other sequence of the oligonucleotide.Moreover, each bead may be linked to oligonucleotides comprising abarcode sequence and an N-mer and may also be linked to a blockeroligonucleotide capable of blocking the oligonucleotides comprising abarcode sequence and an N-mer. Loading of the oligonucleotide blockerand oligonucleotide comprising a barcode sequence and an N-mer may becompleted at distinct ratios in order to obtain desired stoichiometriesof oligonucleotide blocker to oligonucleotide comprising a barcodesequence and an N-mer. In general, a plurality of species may be loadedto beads at distinct ratios in order to obtain desired stoichiometriesof the species on the beads.

Moreover, a bead may also be linked to one or more different types ofmulti-functional oligonucleotides. For example, a multi-functionaloligonucleotide may be capable of functioning as two or more of thefollowing: a primer, a tool for ligation, an oligonucleotide blocker, anoligonucleotide capable of hybridization detection, a reporteroligonucleotide, an oligonucleotide probe, a functional oligonucleotide,an enrichment primer, a targeted primer, a non-specific primer, and afluorescent probe. Oligonucleotides that function as fluorescent probesmay be used, for example, for bead detection or characterization (e.g.,quantification of number of beads, quantification of species (e.g.,primers, linkers, etc.) attached to beads, determination of beadsize/topology, determination of bead porosity, etc.).

Other non-limiting examples of species that may also be attached orcoupled to beads include whole cells, chromosomes, polynucleotides,organic molecules, proteins, polypeptides, carbohydrates, saccharides,sugars, lipids, enzymes, restriction enzymes, ligases, polymerases,barcodes, adapters, small molecules, antibodies, antibody fragments,fluorophores, deoxynucleotide triphosphates (dNTPs), dideoxynucleotidetriphosphates (ddNTPs), buffers, acidic solutions, basic solutions,temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitiveenzymes, metals, metal ions, magnesium chloride, sodium chloride,manganese, aqueous buffer, mild buffer, ionic buffer, inhibitors,saccharides, oils, salts, ions, detergents, ionic detergents, non-ionicdetergents, oligonucleotides, nucleotides, DNA, RNA, peptidepolynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA),single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA,genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA,rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA,ribozyme, riboswitch and viral RNA, a locked nucleic acid (LNA) in wholeor part, locked nucleic acid nucleotides, any other type of nucleic acidanalogue, proteases, nucleases, protease inhibitors, nucleaseinhibitors, chelating agents, reducing agents, oxidizing agents, probes,chromophores, dyes, organics, emulsifiers, surfactants, stabilizers,polymers, water, small molecules, pharmaceuticals, radioactivemolecules, preservatives, antibiotics, aptamers, and combinationsthereof. Both additional oligonucleotide species and other types ofspecies may be coupled to beads by any suitable method includingcovalent and non-covalent means (e.g., ionic bonds, van der Waalsinteractions, hydrophobic interactions, encapsulation, diffusion of thespecies into the bead, etc.). In some cases, an additional species maybe a reactant used for a reaction comprising another type of species onthe bead. For example, an additional species coupled to a bead may be areactant suitable for use in an amplification reaction comprising anoligonucleotide species also attached to the bead.

In some cases, a bead may comprise one or more capture ligands eachcapable of capturing a particular type of sample component, includingcomponents that may comprise nucleic acid. For example, a bead maycomprise a capture ligand capable of capturing a cell from a sample. Thecapture ligand may be, for example, an antibody, antibody fragment,receptor, protein, peptide, small molecule or any other species targetedtoward a species unique to and/or over-expressed on the surface of aparticular cell. Via interactions with the cell target, the particularcell type can be captured from a sample such that it remains bound tothe bead. A bead bound to a cell can be entered into a partition asdescribed elsewhere herein to barcode nucleic acids obtained from thecell. In some cases, capture of a cell from a sample may occur in apartition. Lysis agents, for example, can be included in the partitionsuch in order to release the nucleic acid from the cell. The releasednucleic acid can be barcoded and processed using any of the methodsdescribed herein.

IV. Barcode Libraries

Beads may contain one or more attached barcode sequences. The barcodesequences attached to a single bead may be identical or different. Insome cases, each bead may be attached to about 1, 5, 10, 50, 100, 500,1000, 5000, 10000, 20000, 50000, 100000, 500000, 1000000, 5000000,10000000, 50000000, 100000000, 500000000, 1000000000, 5000000000,10000000000, 50000000000, or 100000000000 identical barcode sequences.In some cases, each bead may be to about 1, 5, 10, 50, 100, 500, 1000,5000, 10000, 20000, 50000, 100000, 500000, 1000000, 5000000, 10000000,50000000, 100000000, 500000000, 1000000000, 5000000000, 10000000000,50000000000, or 100000000000 different barcode sequences. In some cases,each bead may be attached to at least about 1, 5, 10, 50, 100, 500,1000, 5000, 10000, 20000, 50000, 100000, 200000, 300000, 400000, 500000,600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000,5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,30000000, 40000000, 50000000, 60000000, 70000000, 80000000, 90000000,100000000, 200000000, 300000000, 400000000, 500000000, 600000000,700000000, 800000000, 900000000, 1000000000, 2000000000, 3000000000,4000000000, 5000000000, 6000000000, 7000000000, 8000000000, 9000000000,10000000000, 20000000000, 30000000000, 40000000000, 50000000000,60000000000, 70000000000, 80000000000, 90000000000, 100000000000 or moreidentical barcode sequences. In some cases, each bead may be attached toat least about 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000,100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000,1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,9000000, 10000000, 20000000, 30000000, 40000000, 50000000, 60000000,70000000, 80000000, 90000000, 100000000, 200000000, 300000000,400000000, 500000000, 600000000, 700000000, 800000000, 900000000,1000000000, 2000000000, 3000000000, 4000000000, 5000000000, 6000000000,7000000000, 8000000000, 9000000000, 10000000000, 20000000000,30000000000, 40000000000, 50000000000, 60000000000, 70000000000,80000000000, 90000000000, 100000000000 or more different barcodesequences. In some cases, each bead may be attached to less than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000,80000, 90000, 100000, 500000, 1000000, 5000000, 10000000, 50000000,1000000000, 5000000000, 10000000000, 50000000000, or 100000000000identical barcode sequences. In some cases, each bead may be attached toless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000,60000, 70000, 80000, 90000, 100000, 500000, 1000000, 5000000, 10000000,50000000, 1000000000, 5000000000, 10000000000, 50000000000, or100000000000 different barcode sequences.

An individual barcode library may comprise one or more barcoded beads.In some cases, an individual barcode library may comprise about 1, 5,10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 500000,1000000, 5000000, 10000000, 50000000, 100000000, 500000000, 1000000000,5000000000, 10000000000, 50000000000, or 100000000000 individualbarcoded beads. In some cases, each library may comprise at least about1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 200000,300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 30000000, 40000000, 50000000, 60000000, 70000000,80000000, 90000000, 100000000, 200000000, 300000000, 400000000,500000000, 600000000, 700000000, 800000000, 900000000, 1000000000,2000000000, 3000000000, 4000000000, 5000000000, 6000000000, 7000000000,8000000000, 9000000000, 10000000000, 20000000000, 30000000000,40000000000, 50000000000, 60000000000, 70000000000, 80000000000,90000000000, 100000000000 or more individual barcoded beads. In somecases, each library may comprise less than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,500000, 1000000, 5000000, 10000000, 50000000, 1000000000, 5000000000,10000000000, 50000000000, or 100000000000 individual barcoded beads. Thebarcoded beads within the library may have the same sequences ordifferent sequences.

In some embodiments, each bead may have a unique barcode sequence.However, the number of beads with unique barcode sequences within abarcode library may be limited by combinatorial limits. For example,using four different nucleotides, if a barcode is 12 nucleotides inlength, than the number of unique constructs may be limited to4¹²=16777216 unique constructs. Since barcode libraries may comprisemany more beads than 1677216, there may be some libraries with multiplecopies of the same barcode. In some embodiments, the percentage ofmultiple copies of the same barcode within a given library may be 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. Insome cases, the percentage of multiple copies of the same barcode withina given library may be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the percentageof multiple copies of the same barcode within a given library may beless than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, or 50%.

In some embodiments, each bead may comprise one unique barcode sequencebut multiple different random N-mers. In some cases, each bead may haveone or more different random N-mers. Again, the number of beads withdifferent random N-mers within a barcode library may be limited bycombinatorial limits. For example, using four different nucleotides, ifan N-mer sequence is 12 nucleotides in length, than the number ofdifferent constructs may be limited to 4¹²=16777216 differentconstructs. Since barcode libraries may comprise many more beads than16777216, there may be some libraries with multiple copies of the sameN-mer sequence. In some embodiments, the percentage of multiple copiesof the same N-mer sequence within a given library may be 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In some cases,the percentage of multiple copies of the same N-mer sequence within agiven library may be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the percentage ofmultiple copies of the same N-mer sequence within a given library may beless than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, or 50%.

In some embodiments, the unique identifier sequence within the barcodemay be different for each primer within each bead. In some cases, theunique identifier sequence within the barcode sequence may be the samefor each primer within each bead.

V. Combining Barcoded Beads with Sample Types of Samples

The methods, compositions, devices, and kits of this disclosure may beused with any suitable sample or species. A sample (e.g., samplematerial, component of a sample material, fragment of a sample material,etc.) or species can be, for example, any substance used in sampleprocessing, such as a reagent or an analyte. Exemplary samples caninclude one or more of whole cells, chromosomes, polynucleotides,organic molecules, proteins, nucleic acids, polypeptides, carbohydrates,saccharides, sugars, lipids, enzymes, restriction enzymes, ligases,polymerases, barcodes (e.g., including barcode sequences, nucleic acidbarcode sequences, barcode molecules), adaptors, small molecules,antibodies, fluorophores, deoxynucleotide triphosphate (dNTPs),dideoxynucleotide triphosphates (ddNTPs), buffers, acidic solutions,basic solutions, temperature-sensitive enzymes, pH-sensitive enzymes,light-sensitive enzymes, metals, metal ions, magnesium chloride, sodiumchloride, manganese, aqueous buffer, mild buffer, ionic buffer,inhibitors, oils, salts, ions, detergents, ionic detergents, non-ionicdetergents, oligonucleotides, template nucleic acid molecules (e.g.,template oligonucleotides, template nucleic acid sequences), nucleicacid fragments, template nucleic acid fragments (e.g., fragments of atemplate nucleic acid generated from fragmenting a template nucleic acidduring fragmentation, fragments of a template nucleic acid generatedfrom a nucleic acid amplification reaction), nucleotides, DNA, RNA,peptide polynucleotides, complementary DNA (cDNA), double stranded DNA(dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA,chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA(mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, proteases,locked nucleic acids in whole or part, locked nucleic acid nucleotides,nucleases, protease inhibitors, nuclease inhibitors, chelating agents,reducing agents, oxidizing agents, probes, chromophores, dyes, organics,emulsifiers, surfactants, stabilizers, polymers, water, pharmaceuticals,radioactive molecules, preservatives, antibiotics, aptamers, and thelike. In summary, the samples that are used will vary depending on theparticular processing needs.

Samples may be derived from human and non-human sources. In some cases,samples are derived from mammals, non-human mammals, rodents,amphibians, reptiles, dogs, cats, cows, horses, goats, sheep, hens,birds, mice, rabbits, insects, slugs, microbes, bacteria, parasites, orfish. Samples may be derived from a variety of cells, including but notlimited to: eukaryotic cells, prokaryotic cells, fungi cells, heartcells, lung cells, kidney cells, liver cells, pancreas cells,reproductive cells, stem cells, induced pluripotent stem cells,gastrointestinal cells, blood cells, cancer cells, bacterial cells,bacterial cells isolated from a human microbiome sample, etc. In somecases, a sample may comprise the contents of a cell, such as, forexample, the contents of a single cell or the contents of multiplecells. Examples of single cell applications of the methods and systemsdescribed herein are set forth in U.S. Provisional Patent ApplicationNo. 62/017,558 (Attorney Docket No. 43487-728.101), filed of even dateherewith. Samples may also be cell-free, such as circulating nucleicacids (e.g., DNA, RNA).

A sample may be naturally-occurring or synthetic. A sample may beobtained from any suitable location, including from organisms, wholecells, cell preparations and cell-free compositions from any organism,tissue, cell, or environment. A sample may be obtained fromenvironmental biopsies, aspirates, formalin fixed embedded tissues, air,agricultural samples, soil samples, petroleum samples, water samples, ordust samples. In some instances, a sample may be obtained from bodilyfluids, which may include blood, urine, feces, serum, lymph, saliva,mucosal secretions, perspiration, central nervous system fluid, vaginalfluid, or semen. Samples may also be obtained from manufacturedproducts, such as cosmetics, foods, personal care products, and thelike. Samples may be the products of experimental manipulation includingrecombinant cloning, polynucleotide amplification, polymerase chainreaction (PCR) amplification, purification methods (such as purificationof genomic DNA or RNA), and synthesis reactions.

Methods of Attaching Barcodes to Samples

Barcodes (or other oligonucleotides, e.g. random N-mers) may be attachedto a sample by joining the two nucleic acid segments together throughthe action of an enzyme. This may be accomplished by primer extension,polymerase chain reaction (PCR), another type of reaction using apolymerase, or by ligation using a ligase. When the ligation method isused to attach a sample to a barcode, the samples may or may not befragmented prior to the ligation step. In some cases, theoligonucleotides (e.g., barcodes, random N-mers) are attached to asample while the oligonucleotides are still attached to the beads. Insome cases, the oligonucleotides (e.g., barcodes, random N-mers) areattached to a sample after the oligonucleotides are released from thebeads, e.g., by cleavage of the oligonucleotides comprising the barcodesfrom the beads and/or through degradation of the beads.

The oligonucleotides may include one or more random N-mer sequences. Acollection of unique random N-mer sequences may prime random portions ofa DNA segment, thereby amplifying a sample (e.g., a whole genome). Theresulting product may be a collection of barcoded fragmentsrepresentative of the entire sample (e.g., genome).

The samples may or may not be fragmented before ligation to barcodedbeads. DNA fragmentation may involve separating or disrupting DNAstrands into small pieces or segments. A variety of methods may beemployed to fragment DNA including restriction digest or various methodsof generating shear forces. Restriction digest may utilize restrictionenzymes to make intentional cuts in a DNA sequence by blunt cleavage toboth strands or by uneven cleavage to generate sticky ends. Examples ofshear-force mediated DNA strand disruption may include sonication,acoustic shearing, needle shearing, pipetting, or nebulization.Sonication, is a type of hydrodynamic shearing, exposing DNA sequencesto short periods of shear forces, which may result in about 700 bpfragment sizes. Acoustic shearing applies high-frequency acoustic energyto the DNA sample within a bowl-shaped transducer. Needle shearinggenerates shear forces by passing DNA through a small diameter needle tophysically tear DNA into smaller segments. Nebulization forces may begenerated by sending DNA through a small hole of an aerosol unit inwhich resulting DNA fragments are collected from the fine mist exitingthe unit.

In some cases, a ligation reaction is used to ligate oligonucleotides tosample. The ligation may involve joining together two nucleic acidsegments, such as a barcode sequence and a sample, by catalyzing theformation of a phosphodiester bond. The ligation reaction may include aDNA ligase, such as an E. coli DNA ligase, a T4 DNA ligase, a mammalianligase such as DNA ligase I, DNA ligase III, DNA ligase IV, thermostableligases, or the like. The T4 DNA ligase may ligate segments containingDNA, oligonucleotides, RNA, and RNA-DNA hybrids. The ligation reactionmay not include a DNA ligase, utilizing an alternative such as atopoisomerase. To ligate a sample to a barcode sequence, utilizing ahigh DNA ligase concentration and including PEG may achieve rapidligation. The optimal temperature for DNA ligase, which may be 37° C.,and the melting temperature of the DNA to be ligated, which may vary,may be considered to select for a favorable temperature for the ligationreaction. The sample and barcoded beads may be suspended in a buffer tominimize ionic effects that may affect ligation.

Although described in terms of ligation or direct attachment of abarcode sequence to a sample nucleic acid component, above, theattachment of a barcode to a sample nucleic acid, as used herein, alsoencompasses the attachment of a barcode sequence to a complement of asample, or a copy or complement of that complement, e.g., when thebarcode is associated with a primer sequence that is used to replicatethe sample nucleic acid, as is described in greater detail elsewhereherein. In particular, where a barcode containing primer sequence isused in a primer extension reaction using the sample nucleic acid (or areplicate of the sample nucleic acid) as a template, the resultingextension product, whether a complement of the sample nucleic acid or aduplicate of the sample nucleic acid, will be referred to as having thebarcode sequence attached to it.

In some cases, sample is combined with the barcoded beads (eithermanually or with the aid of a microfluidic device) and the combinedsample and beads are partitioned, such as in a microfluidic device. Thepartitions may be aqueous droplets within a water-in-oil emulsion. Whensamples are combined with barcoded beads, on average less than twotarget analytes may be present in each fluidic droplet. In someembodiments, on average, less than three target analytes may appear perfluidic droplet. In some cases, on average, more than two targetanalytes may appear per fluidic droplet. In other cases, on average,more than three target analytes may appear per fluidic droplet. In somecases, one or more strands of the same target analyte may appear in thesame fluidic droplet. In some cases, less than 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 50, 100, 1000, 5000, 10000, or 100000 target analytes are presentwithin a fluidic droplet. In some cases, greater than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 50, 100, 1000, 5000, 10000, or 100000 target analytes arepresent within a fluidic droplet. The partitions described herein areoften characterized by having extremely small volumes. For example, inthe case of droplet based partitions, the droplets may have overallvolumes that are less than 1000 pL, less than 900 pL, less than 800 pL,less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL,less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL,less than 20 pL, less than 10 pL, or even less than 1 pL. Whereco-partitioned with beads, it will be appreciated that the sample fluidvolume within the partitions may be less than 90% of the above describedvolumes, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, or even less than 10% theabove described volumes.

When samples are combined with barcoded beads, on average less than onebead may be present in each fluidic droplet. In some embodiments, onaverage, less than two beads may be present in each fluidic droplet. Insome embodiments, on average, less than three beads may be present perfluidic droplet. In some cases, on average, more than one bead may bepresent in each fluidic droplet. In other cases, on average, more thantwo beads may appear be present in each fluidic droplet. In other cases,on average, more than three beads may be present per fluidic droplet. Insome embodiments, a ratio of on average less than one barcoded bead perfluidic droplet may be achieved using limiting dilution technique. Here,barcoded beads may be diluted prior to mixing with the sample, dilutedduring mixing with the sample, or diluted after mixing with the sample.

The number of different barcodes or different sets of barcodes (e.g.,different sets of barcodes, each different set coupled to a differentbead) that are partitioned may vary depending upon, for example, theparticular barcodes to be partitioned and/or the application. Differentsets of barcodes may be, for example, sets of identical barcodes wherethe identical barcodes differ between each set. Or different sets ofbarcodes may be, for example, sets of different barcodes, where each setdiffers in its included barcodes. In some cases, different barcodes arepartitioned by attaching different barcodes to different beads (e.g.,gel beads). In some cases, different sets of barcodes are partitioned bydisposing each different set in a different partition. In some cases,though a partition may comprise one or more different barcode sets. Forexample, each different set of barcodes may be coupled to a differentbead (e.g., a gel bead). Each different bead may be partitioned into afluidic droplet, such that each different set of barcodes is partitionedinto a different fluidic droplet. For example, about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, ormore different barcodes or different sets of barcodes may bepartitioned. In some examples, at least about 1, 5, 10, 50, 100, 1000,10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,9000000, 10000000, 20000000, 50000000, 100000000, or more differentbarcodes or different sets of barcodes may be partitioned. In someexamples, less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000,40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000,3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 different barcodes or different sets ofbarcodes may be partitioned. In some examples, about 1-5, 5-10, 10-50,50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 different barcodes ordifferent sets of barcodes may be partitioned.

Barcodes may be partitioned at a particular density. For example,barcodes may be partitioned so that each partition contains about 1, 5,10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000,600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000,5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,50000000, or 100000000 barcodes per partition. Barcodes may bepartitioned so that each partition contains at least about 1, 5, 10, 50,100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, ormore barcodes per partition. Barcodes may be partitioned so that eachpartition contains less than about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, or 100000000 barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 barcodes perpartition. In some cases, partitioned barcodes may be coupled to one ormore beads, such as, for example, a gel bead. In some cases, thepartitions are fluidic droplets.

Barcodes may be partitioned such that identical barcodes are partitionedat a particular density. For example, identical barcodes may bepartitioned so that each partition contains about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000identical barcodes per partition. Barcodes may be partitioned so thateach partition contains at least about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, 100000000, or more identical barcodes perpartition. Barcodes may be partitioned so that each partition containsless than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000,4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 identical barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 identical barcodes perpartition. In some cases, partitioned identical barcodes may be coupledto a bead, such as, for example, a gel bead. In some cases, thepartitions are fluidic droplets.

Barcodes may be partitioned such that different barcodes are partitionedat a particular density. For example, different barcodes may bepartitioned so that each partition contains about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000different barcodes per partition. Barcodes may be partitioned so thateach partition contains at least about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, 100000000, or more different barcodes perpartition. Barcodes may be partitioned so that each partition containsless than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000,4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 different barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 different barcodes perpartition. In some cases, partitioned different barcodes may be coupledto a bead, such as, for example, a gel bead. In some cases, thepartitions are fluidic droplets.

The number of partitions employed to partition barcodes or differentsets of barcodes may vary, for example, depending on the applicationand/or the number of different barcodes or different sets of barcodes tobe partitioned. For example, the number of partitions employed topartition barcodes or different sets of barcodes may be about 5, 10, 50,100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000,300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000,2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000 or more.The number of partitions employed to partition barcodes or differentsets of barcodes may be at least about 5, 10, 50, 100, 250, 500, 750,1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000,60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000,600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000,5000000, 10000000, 20000000 or more. The number of partitions employedto partition barcodes or different sets of barcodes may be less thanabout 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500,10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,2000000, 3000000, 4000000, 5000000, 10000000, or 20000000. The number ofpartitions employed to partition barcodes may be about 5-10000000,5-5000000, 5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000,1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000. In some cases,the partitions may be fluidic droplets.

As described above, different barcodes or different sets of barcodes(e.g., each set comprising a plurality of identical barcodes ordifferent barcodes) may be partitioned such that each partitiongenerally comprises a different barcode or different barcode set. Insome cases, each partition may comprise a different set of identicalbarcodes, such as an identical set of barcodes coupled to a bead (e.g.,a gel bead). Where different sets of identical barcodes are partitioned,the number of identical barcodes per partition may vary. For example,about 100,000 or more different sets of identical barcodes (e.g., a setof identical barcodes attached to a bead) may be partitioned acrossabout 100,000 or more different partitions, such that each partitioncomprises a different set of identical barcodes (e.g., each partitioncomprises a bead coupled to a different set of identical barcodes). Ineach partition, the number of identical barcodes per set of barcodes maybe about 1,000,000 or more identical barcodes (e.g., each partitioncomprises 1,000,000 or more identical barcodes coupled to one or morebeads). In some cases, the number of different sets of barcodes may beequal to or substantially equal to the number of partitions or may beless than the number of partitions. Any suitable number of differentbarcodes or different barcode sets, number of barcodes per partition,and number of partitions may be combined. Thus, as will be appreciated,any of the above-described different numbers of barcodes may be providedwith any of the above-described barcode densities per partition, and inany of the above-described numbers of partitions.

Microfluidic Devices and Droplets

In some cases, this disclosure provides devices for making beads and forcombining beads (or other types of partitions) with samples, e.g., forco-partitioning sample components and beads. Such a device may be amicrofluidic device (e.g., a droplet generator). The device may beformed from any suitable material. In some examples, a device may beformed from a material selected from the group consisting of fusedsilica, soda lime glass, borosilicate glass, poly(methyl methacrylate)PMMA, PDMS, sapphire, silicon, germanium, cyclic olefin copolymer,polyethylene, polypropylene, polyacrylate, polycarbonate, plastic,thermosets, hydrogels, thermoplastics, paper, elastomers, andcombinations thereof.

A device may be formed in a manner that it comprises channels for theflow of fluids. Any suitable channels may be used. In some cases, adevice comprises one or more fluidic input channels (e.g., inletchannels) and one or more fluidic outlet channels. In some embodiments,the inner diameter of a fluidic channel may be about 10 μm, 20 μm, 100μm, 125 μm, or 150 μm. In some cases, the inner diameter of a fluidicchannel may be more than 10 μm, 100 μm, 125 μm, 150 μm or more. In someembodiments, the inner diameter of a fluidic channel may be less thanabout 10 μm, 20 μm, 100 μm, 125 μm, or 150 μm. Volumetric flow rateswithin a fluidic channel may be any flow rate known in the art.

As described elsewhere herein, the microfluidic device may be utilizedto form beads by forming a fluidic droplet comprising one or more gelprecursors, one or more crosslinkers, optionally an initiator, andoptionally an aqueous surfactant. The fluidic droplet may be surroundedby an immiscible continuous fluid, such as an oil, which may furthercomprise a surfactant and/or an accelerator.

In some embodiments, the microfluidic device may be used to combinebeads (e.g., barcoded beads or other type of first partition, includingany suitable type of partition described herein) with sample (e.g., asample of nucleic acids) by forming a fluidic droplet (or other type ofsecond partition, including any suitable type of partition describedherein) comprising both the beads and the sample. The fluidic dropletmay have an aqueous core surrounded by an oil phase, such as, forexample, aqueous droplets within a water-in-oil emulsion. The fluidicdroplet may contain one or more barcoded beads, a sample, amplificationreagents, and a reducing agent. In some cases, the fluidic droplet mayinclude one or more of water, nuclease-free water, acetonitrile, beads,gel beads, polymer precursors, polymer monomers, polyacrylamidemonomers, acrylamide monomers, degradable crosslinkers, non-degradablecrosslinkers, disulfide linkages, acrydite moieties, PCR reagents,primers, polymerases, barcodes, polynucleotides, oligonucleotides,nucleotides, DNA, RNA, peptide polynucleotides, complementary DNA(cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA),plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA,bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA,snRNA, snoRNA, scaRNA, microRNA, dsRNA, probes, dyes, organics,emulsifiers, surfactants, stabilizers, polymers, aptamers, reducingagents, initiators, biotin labels, fluorophores, buffers, acidicsolutions, basic solutions, light-sensitive enzymes, pH-sensitiveenzymes, aqueous buffer, oils, salts, detergents, ionic detergents,non-ionic detergents, and the like. In summary, the composition of thefluidic droplet will vary depending on the particular processing needs.

The fluidic droplets may be of uniform size or heterogeneous size. Insome cases, the diameter of a fluidic droplet may be about 1 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm. In some cases, a fluidicdroplet may have a diameter of at least about 1 μm, 5 μm, 10 μm, 20 μm,30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm,100 μm, 250 μm, 500 μm, 1 mm or more. In some cases, a fluidic dropletmay have a diameter of less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm,40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm,250 μm, 500 μm, or 1 mm. In some cases, fluidic droplet may have adiameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm,40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

In some embodiments, the device may comprise one or more intersectionsof two or more fluid input channels. For example, the intersection maybe a fluidic cross. The fluidic cross may comprise two or more fluidicinput channels and one or more fluidic outlet channels. In some cases,the fluidic cross may comprise two fluidic input channels and twofluidic outlet channels. In other cases, the fluidic cross may comprisethree fluidic input channels and one fluidic outlet channel. In somecases, the fluidic cross may form a substantially perpendicular anglebetween two or more of the fluidic channels forming the cross.

In some cases, a microfluidic device may comprise a first and a secondinput channel that meet at a junction that is fluidly connected to anoutput channel. In some cases, the output channel may be, for example,fluidly connected to a third input channel at a junction. In some cases,a fourth input channel may be included and may intersect the third inputchannel and outlet channel at a junction. In some cases, a microfluidicdevice may comprise first, second, and third input channels, wherein thethird input channel intersects the first input channel, the second inputchannel, or a junction of the first input channel and the second inputchannel.

As described elsewhere herein, the microfluidic device may be used togenerate gel beads from a liquid. For example, in some embodiments, anaqueous fluid comprising one or more gel precursors, one or morecrosslinkers and optionally an initiator, optionally an aqueoussurfactant, and optionally an alcohol within a fluidic input channel mayenter a fluidic cross. Within a second fluidic input channel, an oilwith optionally a surfactant and an accelerator may enter the samefluidic cross. Both aqueous and oil components may be mixed at thefluidic cross causing aqueous fluidic droplets to form within thecontinuous oil phase. Gel precursors within fluidic droplets exiting thefluidic cross may polymerize forming beads.

As described elsewhere herein, the microfluidic device (e.g., a dropletgenerator) may be used to combine sample with beads (e.g., a library ofbarcoded beads) as well as an agent capable of degrading the beads(e.g., reducing agent if the beads are linked with disulfide bonds), ifdesired. In some embodiments, a sample (e.g., a sample of nucleic acids)may be provided to a first fluidic input channel that is fluidlyconnected to a first fluidic cross (e.g., a first fluidic junction).Pre-formed beads (e.g., barcoded beads, degradable barcoded beads) maybe provided to a second fluidic input channel that is also fluidlyconnected to the first fluidic cross, where the first fluidic inputchannel and second fluidic input channel meet. The sample and beads maybe mixed at the first fluidic cross to form a mixture (e.g., an aqueousmixture). In some cases, a reducing agent may be provided to a thirdfluidic input channel that is also fluidly connected to the firstfluidic cross and meets the first and second fluidic input channel atthe first fluidic cross. The reducing agent can then be mixed with thebeads and sample in the first fluidic cross. In other cases, thereducing agent may be premixed with the sample and/or the beads beforeentering the microfluidic device such that it is provided to themicrofluidic device through the first fluidic input channel with thesample and/or through the second fluidic input channel with the beads.In other cases, no reducing agent may be added.

In some embodiments, the sample and bead mixture may exit the firstfluidic cross through a first outlet channel that is fluidly connectedto the first fluidic cross (and, thus, any fluidic channels forming thefirst fluidic cross). The mixture may be provided to a second fluidiccross (e.g., a second fluidic junction) that is fluidly connected to thefirst outlet channel. In some cases, an oil (or other suitableimmiscible) fluid may enter the second fluidic cross from one or moreseparate fluidic input channels that are fluidly connected to the secondfluidic cross (and, thus, any fluidic channels forming the cross) andthat meet the first outlet channel at the second fluidic cross. In somecases, the oil (or other suitable immiscible fluid) may be provided inone or two separate fluidic input channels fluidly connected to thesecond fluidic cross (and, thus, the first outlet channel) that meet thefirst outlet channel and each other at the second fluidic cross. Bothcomponents, the oil and the sample and bead mixture, may be mixed at thesecond fluidic cross. This mixing partitions the sample and bead mixtureinto a plurality of fluidic droplets (e.g., aqueous droplets within awater-in-oil emulsion), in which at least a subset of the droplets thatform encapsulate a barcoded bead (e.g., a gel bead). The fluidicdroplets that form may be carried within the oil through a secondfluidic outlet channel exiting from the second fluidic cross. In somecases, fluidic droplets exiting the second outlet channel from thesecond fluidic cross may be partitioned into wells for furtherprocessing (e.g., thermocycling).

In many cases, it will be desirable to control the occupancy rate ofresulting droplets (or second partitions) with respect to beads (orfirst partitions). Such control is described in, for example, U.S.Provisional patent application No. 61/977,804, filed Apr. 4, 2014, thefull disclosure of which is incorporated herein by reference in itsentirety for all purposes. In general, the droplets (or secondpartitions) will be formed such that at least 50%, 60%, 70%, 80%, 90% ormore droplets (or second partitions) contain no more than one bead (orfirst partition). Additionally, or alternatively, the droplets (orsecond partitions) will be formed such that at least 50%, 60%, 70%, 80%,90% or more droplets (or second partitions) include exactly one bead (orfirst partition). In some cases, the resulting droplets (or secondpartitions) may each comprise, on average, at most about one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, ortwenty beads (or first partitions). In some cases, the resultingdroplets (or second partitions) may each comprise, on average, at leastabout one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty, or more beads (or first partitions).

In some embodiments, samples may be pre-mixed with beads (e.g.,degradable beads) comprising barcodes and any other reagent (e.g.,reagents necessary for sample amplification, a reducing agent, etc.)prior to entry of the mixture into a microfluidic device to generate anaqueous reaction mixture. Upon entry of the aqueous mixture to a fluidicdevice, the mixture may flow from a first fluidic input channel and intoa fluidic cross. In some cases, an oil phase may enter the fluidic crossfrom a second fluidic input channel (e.g., a fluidic channelperpendicular to or substantially perpendicular to the first fluidicinput channel) also fluidly connected to the fluidic cross. The aqueousmixture and oil may be mixed at the fluidic cross, such that an emulsion(e.g. a gel-water-oil emulsion) forms. The emulsion can comprise aplurality of fluidic droplets (e.g., droplets comprising the aqueousreaction mixture) in the continuous oil phase. In some cases, eachfluidic droplet may comprise a single bead (e.g., a gel bead attached toa set of identical barcodes), an aliquot of sample, and an aliquot ofany other reagents (e.g., reducing agents, reagents necessary foramplification of the sample, etc.). In some cases, though, a fluidicdroplet may comprise a plurality of beads. Upon droplet formation, thedroplet may be carried via the oil continuous phase through a fluidicoutlet channel exiting from the fluidic cross. Fluidic droplets exitingthe outlet channel may be partitioned into wells for further processing(e.g., thermocycling).

In cases where a reducing agent may be added to the sample prior toentering the microfluidic device or may be added at the first fluidiccross, the fluidic droplets formed at the second fluidic cross maycontain the reducing agent. In this case, the reducing agent may degradeor dissolve the beads contained within the fluidic droplet as thedroplet travels through the outlet channel leaving the second fluidiccross.

In some embodiments, a microfluidic device may contain three discretefluidic crosses in parallel. Fluidic droplets may be formed at any oneof the three fluidic crosses. Sample and beads may be combined withinany one of the three fluidic crosses. A reducing agent may be added atany one of the three fluidic crosses. An oil may be added at any one ofthe three fluidic crosses.

The methods, compositions, devices, and kits of this disclosure may beused with any suitable oil. In some embodiments, an oil may be used togenerate an emulsion. The oil may comprise fluorinated oil, silicon oil,mineral oil, vegetable oil, and combinations thereof.

In some embodiments, the aqueous fluid within the microfluidic devicemay also contain an alcohol. For example, an alcohol may be glycerol,ethanol, methanol, isopropyl alcohol, pentanol, ethane, propane, butane,pentane, hexane, and combinations thereof. The alcohol may be presentwithin the aqueous fluid at about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (v/v). In some cases, thealcohol may be present within the aqueous fluid at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% ormore (v/v). In some cases, the alcohol may be present within the aqueousfluid for less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, or 20% (v/v).

In some embodiments, the oil may also contain a surfactant to stabilizethe emulsion. For example, a surfactant may be a fluorosurfactant,Krytox lubricant, Krytox FSH, an engineered fluid, HFE-7500, a siliconecompound, a silicon compound containing PEG, such as bis krytox peg(BKP). The surfactant may be present at about 0.1%, 0.5%, 1%, 1.1%,1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 5%, or 10% (w/w). Insome cases, the surfactant may be present at least about 0.1%, 0.5%, 1%,1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 5%, 10% (w/w)or more. In some cases, the surfactant may be present for less thanabout 0.1%, 0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2%, 5%, or 10% (w/w).

In some embodiments, an accelerator and/or initiator may be added to theoil. For example, an accelerator may be Tetramethylethylenediamine(TMEDA or TEMED). In some cases, an initiator may be ammonium persulfateor calcium ions. The accelerator may be present at about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% (v/v). In some cases, theaccelerator may be present at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,1.8%, 1.9%, or 2% (v/v) or more. In some cases, the accelerator may bepresent for less than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or2% (v/v).

VI. Amplification

DNA amplification is a method for creating multiple copies of small orlong segments of DNA. The methods, compositions, devices, and kits ofthis disclosure may use DNA amplification to attach one or more desiredoligonucleotide sequences to individual beads, such as a barcodesequence or random N-mer sequence. DNA amplification may also be used toprime and extend along a sample of interest, such as genomic DNA,utilizing a random N-mer sequence, in order to produce a fragment of thesample sequence and couple the barcode associated with the primer tothat fragment.

For example, a nucleic acid sequence may be amplified by co-partitioninga template nucleic acid sequence and a bead comprising a plurality ofattached oligonucleotides (e.g., releasably attached oligonucleotides)into a partition (e.g., a droplet of an emulsion, a microcapsule, or anyother suitable type of partition, including a suitable type of partitiondescribed elsewhere herein). The attached oligonucleotides can comprisea primer sequence (e.g., a variable primer sequence such as, forexample, a random N-mer, or a targeted primer sequence such as, forexample, a targeted N-mer) that is complementary to one or more regionsof the template nucleic acid sequence and, in addition, may alsocomprise a common sequence (e.g., such as a barcode sequence). Theprimer sequence can be annealed to the template nucleic acid sequenceand extended (e.g., in a primer extension reaction or any other suitablenucleic acid amplification reaction) to produce one or more first copiesof at least a portion of the template nucleic acid, such that the one ormore first copies comprises the primer sequence and the common sequence.In cases where the oligonucleotides comprising the primer sequence arereleasably attached to the bead, the oligonucleotides may be releasedfrom the bead prior to annealing the primer sequence to the templatenucleic acid sequence. Moreover, in general, the primer sequence may beextended via a polymerase enzyme (e.g., a strand displacing polymeraseenzyme as described elsewhere herein, an exonuclease deficientpolymerase enzyme as described elsewhere herein, or any other type ofsuitable polymerase, including a type of polymerase described elsewhereherein) that is also provided in the partition. Furthermore, theoligonucleotides releasably attached to the bead may be exonucleaseresistant and, thus, may comprise one or more phosphorothioate linkagesas described elsewhere herein. In some cases, the one or morephosphorothioate linkages may comprise a phosphorothioate linkage at aterminal internucleotide linkage in the oligonucleotides.

In some cases, after the generation of the one or more first copies, theprimer sequence can be annealed to one or more of the first copies andthe primer sequence again extended to produce one or more second copies.The one or more second copies can comprise the primer sequence, thecommon sequence, and may also comprise a sequence complementary to atleast a portion of an individual copy of the one or more first copies,and/or a sequence complementary to the variable primer sequence. Theaforementioned steps may be repeated for a desired number of cycles toproduce amplified nucleic acids.

The oligonucleotides described above may comprise a sequence segmentthat is not copied during an extension reaction (such as an extensionreaction that produces the one or more first or second copies describedabove). As described elsewhere herein, such a sequence segment maycomprise one or more uracil containing nucleotides and may also resultin the generation of amplicons that form a hairpin (or partial hairpin)molecule under annealing conditions.

In another example, a plurality of different nucleic acids can beamplified by partitioning the different nucleic acids into separatefirst partitions (e.g., droplets in an emulsion) that each comprise asecond partition (e.g., beads, including a type of bead describedelsewhere herein). The second partition may be releasably associatedwith a plurality of oligonucleotides. The second partition may compriseany suitable number of oligonucleotides (e.g., more than 1,000oligonucleotides, more than 10,000 oligonucleotides, more than 100,000oligonucleotides, more than 1,000,000 oligonucleotides, more than10,000,000 oligonucleotides, or any other number of oligonucleotides perpartition described herein). Moreover, the second partitions maycomprise any suitable number of different barcode sequences (e.g., atleast 1,000 different barcode sequences, at least 10,000 differentbarcode sequences, at least 100,000 different barcode sequences, atleast 1,000,000 different barcode sequences, at least 10,000,000different barcode sequence, or any other number of different barcodesequences described elsewhere herein).

Furthermore, the plurality of oligonucleotides associated with a givensecond partition may comprise a primer sequence (e.g., a variable primersequence, a targeted primer sequence) and a common sequence (e.g., abarcode sequence). Moreover, the plurality of oligonucleotidesassociated with different second partitions may comprise differentbarcode sequences. Oligonucleotides associated with the plurality ofsecond partitions may be released into the first partitions. Followingrelease, the primer sequences within the first partitions can beannealed to the nucleic acids within the first partitions and the primersequences can then be extended to produce one or more copies of at leasta portion of the nucleic acids with the first partitions. In general,the one or more copies may comprise the barcode sequences released intothe first partitions.

Amplification Within Droplets and Sample Indexing

Nucleic acid (e.g., DNA) amplification may be performed on contentswithin fluidic droplets. As described herein, fluidic droplets maycontain oligonucleotides attached to beads. Fluidic droplets may furthercomprise a sample. Fluidic droplets may also comprise reagents suitablefor amplification reactions which may include Kapa HiFi Uracil Plus,modified nucleotides, native nucleotides, uracil containing nucleotides,dTTPs, dUTPs, dCTPs, dGTPs, dATPs, DNA polymerase, Taq polymerase,mutant proof reading polymerase, 9 degrees North, modified (NEB), exo(-), exo (-) Pfu, Deep Vent exo (-), Vent exo (-), and acyclonucleotides(acyNTPS).

Oligonucleotides attached to beads within a fluidic droplet may be usedto amplify a sample nucleic acid such that the oligonucleotides becomeattached to the sample nucleic acid. The sample nucleic acids maycomprise virtually any nucleic acid sought to be analyzed, including,for example, whole genomes, exomes, amplicons, targeted genome segmentse.g., genes or gene families, cellular nucleic acids, circulatingnucleic acids, and the like, and, as noted above, may include DNA(including gDNA, cDNA, mtDNA, etc.) RNA (e.g., mRNA, rRNA, total RNA,etc.). Preparation of such nucleic acids for barcoding may generally beaccomplished by methods that are readily available, e.g., enrichment orpull-down methods, isolation methods, amplification methods etc. Inorder to amplify a desired sample, such as gDNA, the random N-mersequence of an oligonucleotide within the fluidic droplet may be used toprime the desired target sequence and be extended as a complement of thetarget sequence. In some cases, the oligonucleotide may be released fromthe bead in the droplet, as described elsewhere herein, prior topriming. For these priming and extension processes, any suitable methodof DNA amplification may be utilized, including polymerase chainreaction (PCR), digital PCR, reverse-transcription PCR, multiplex PCR,nested PCR, overlap-extension PCR, quantitative PCR, multipledisplacement amplification (MDA), or ligase chain reaction (LCR). Insome cases, amplification within fluidic droplets may be performed untila certain amount of sample nucleic acid comprising barcode may beproduced. In some cases, amplification may be performed for about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20cycles. In some cases, amplification may be performed for more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 cycles, or more. In some cases, amplification may be performed forless than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 cycles.

An exemplary amplification and barcoding process as described herein, isschematically illustrated in FIGS. 38A-38F. As shown, oligonucleotidesthat include a barcode sequence are co-partitioned in, e.g., a droplet3802 in an emulsion, along with a sample nucleic acid 3804. As notedelsewhere herein, the oligonucleotides 3808 may be provided on a bead3806 that is co-partitioned with the sample nucleic acid 3804, whicholigonucleotides are preferably releasable from the bead 3806, as shownin FIG. 38A. The oligonucleotides 3808 include a barcode sequence 3812,in addition to one or more functional sequences, e.g., sequences 3810,3814 and 3816. For example, oligonucleotide 3808 is shown as comprisingbarcode sequence 3812, as well as sequence 3810 that may function as anattachment or immobilization sequence for a given sequencing system,e.g., a P5 sequence used for attachment in flow cells of an IlluminaHiseq or Miseq system. As shown, the oligonucleotides also include aprimer sequence 3816, which may include a random or targeted N-mer forpriming replication of portions of the sample nucleic acid 3804. Alsoincluded within oligonucleotide 3808 is a sequence 3814 which mayprovide a sequencing priming region, such as a “read1” or R1 primingregion, that is used to prime polymerase mediated, template directedsequencing by synthesis reactions in sequencing systems. In many cases,the barcode sequence 3812, immobilization sequence 3810 and R1 sequence3814 will be common to all of the oligonucleotides attached to a givenbead. The primer sequence 3816 may vary for random N-mer primers, or maybe common to the oligonucleotides on a given bead for certain targetedapplications.

Based upon the presence of primer sequence 3816, the oligonucleotidesare able to prime the sample nucleic acid as shown in FIG. 38B, whichallows for extension of the oligonucleotides 3808 and 3808 a usingpolymerase enzymes and other extension reagents also co-partitioned withthe bead 3806 and sample nucleic acid 3804. As described elsewhereherein, these polymerase enzymes may include thermostable polymerases,e.g., where initial denaturation of double stranded sample nucleic acidswithin the partitions is desired. Alternatively, denaturation of samplenucleic acids may precede partitioning, such that single stranded targetnucleic acids are deposited into the partitions, allowing the use ofnon-thermostable polymerase enzymes, e.g., Klenow, phi29, Pol 1, and thelike, where desirable. As shown in FIG. 38C, following extension of theoligonucleotides that, for random N-mer primers, would anneal tomultiple different regions of the sample nucleic acid 3804; multipleoverlapping complements or fragments of the nucleic acid are created,e.g., fragments 3818 and 3820. Although including sequence portions thatare complementary to portions of sample nucleic acid, e.g., sequences3822 and 3824, these constructs are generally referred to herein ascomprising fragments of the sample nucleic acid 3804, having theattached barcode sequences. In some cases, it may be desirable toartificially limit the size of the replicate fragments that are producedin order to maintain manageable fragment sizes from the firstamplification steps. In some cases, this may be accomplished bymechanical means, as described above, e.g., using fragmentation systemslike a Covaris system, or it may be accomplished by incorporating randomextension terminators, e.g., at low concentrations, to prevent theformation of excessively long fragments.

These fragments may then be subjected to sequence analysis, or they maybe further amplified in the process, as shown in FIG. 38D. For example,additional oligonucleotides, e.g., oligonucleotide 3808 b, also releasedfrom bead 3806, may prime the fragments 3818 and 3820. This shown in forfragment 3818. In particular, again, based upon the presence of therandom N-mer primer 3816 b in oligonucleotide 3808 b (which in manycases will be different from other random N-mers in a given partition,e.g., primer sequence 3816), the oligonucleotide anneals with thefragment 3818, and is extended to create a complement 3826 to at least aportion of fragment 3818 which includes sequence 3828, that comprises aduplicate of a portion of the sample nucleic acid sequence. Extension ofthe oligonucleotide 3808 b continues until it has replicated through theoligonucleotide portion 3808 of fragment 3818. As noted elsewhereherein, and as illustrated in panel D, the oligonucleotides may beconfigured to prompt a stop in the replication by the polymerase at adesired point, e.g., after replicating through sequences 3816 and 3814of oligonucleotide 3808 that is included within fragment 3818. Asdescribed herein, this may be accomplished by different methods,including, for example, the incorporation of different nucleotidesand/or nucleotide analogues that are not capable of being processed bythe polymerase enzyme used. For example, this may include the inclusionof uracil containing nucleotides within the sequence region 3812 tocause a non-uracil tolerant polymerase to cease replication of thatregion. As a result, a fragment 3826 is created that includes thefull-length oligonucleotide 3808 b at one end, including the barcodesequence 3812, the attachment sequence 3810, the R1 primer region 3814,and the random n-mer sequence 3816 b. At the other end of the sequencewill be included the complement 3816′ to the random n-mer of the firstoligonucleotide 3808, as well as a complement to all or a portion of theR1 sequence, shown as sequence 3814′. The R1 sequence 3814 and itscomplement 3814′ are then able to hybridize together to form a partialhairpin structure 3828. As will be appreciated because the random-n-mersdiffer among different oligonucleotides, these sequences and theircomplements would not be expected to participate in hairpin formation,e.g., sequence 3816′, which is the complement to random N-mer 3816,would not be expected to be complementary to random n-mer sequence 3816b. This would not be the case for other applications, e.g., targetedprimers, where the N-mers may be common among oligonucleotides within agiven partition.

By forming these partial hairpin structures, it allows for the removalof first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 3826.

Following attachment of the barcode to the sample, additionalamplification steps (e.g. PCR) may be performed to amplify the barcodedfragments prior to sequencing, as well as to optionally add additionalfunctional sequences to those barcoded fragments, e.g., additionalprimer binding sites (e.g. Read2 sequence primer, Index primer) that iscompatible with a sequencing device (e.g. Illumina MiSeq) andoptionally, one or more additional barcode sequences (e.g., see FIG.14C), as well as other functional sequences, e.g., additionalimmobilization sequences or their complements, e.g., P7 sequences. Insome cases, an additional barcode sequence may serve as a sample index,with the original barcode and sample index permitting multiplexedsequencing (e.g., simultaneous molecular tagging and sampleidentification). The original barcode can be used during sequencing toalign a sequence read corresponding to the nucleic acid moleculeassociated with the barcode (e.g., identified via the barcode). Adifferent sample index can be included in sequencer-ready productsgenerated from each different sample. Thus, the sample index can be usedduring sequencing for identifying the sample to which a particularsequence read belongs and multiplexing can be achieved.

In some cases, a sample index can be added to a sample nucleic acidafter the addition of the original barcode to the sample nucleic acid,with or without the use of partitions or the generation of additionalpartitions. In some cases, the sample index is added in bulk. In somecases, the addition of a sample index to a sample nucleic acid may occurprior to the addition of a barcode to the sample nucleic acid. In somecases, the addition of a sample index to a sample nucleic acid may occursimultaneous to or in parallel to the addition of a sample index to thesample nucleic acid.

In some cases, a sample index may be added to a sample nucleic acidafter addition of a barcode sequence to the sample nucleic acid. Forexample, as described elsewhere herein, amplification methods may beused to attach a barcode sequence and other sequences (e.g., P5, R1,etc.) to a sample nucleic acid. In some cases, a random amplificationscheme, such as Partial Hairpin Amplification for Sequencing (PHASE—asdescribed elsewhere herein), for example, may aid in attaching a barcodesequence and other sequences to a sample nucleic acid. In one example, aplurality of primers, each comprising a different random N-mer, asequencer attachment or immobilization site (e.g., P5), a barcodesequence (e.g., an identical barcode sequence), and a sequencing primerbinding site (e.g., R1) are used to randomly prime and amplify a samplenucleic acid. Any of the sequencer primer binding site, the barcodesequence, and/or sequencing primer binding site may comprise uracilcontaining nucleotides. The primer may also include an oligonucleotideblocker hybridized to the primer at one or more sequences of the primerto ensure that priming of the sample nucleic acid occurs only via therandom N-mer. A schematic representation of an example primer is asfollows (oligonucleotide blocker not shown):

-   P5-Barcode-R1-RandomNMer

Random priming of the sample nucleic acid and multiple rounds ofamplification can generate amplicons comprising a portion of the samplenucleic acid linked at one end to the sequencer attachment orimmobilization site (e.g., P5), the barcode, the sequencing primerbinding site (e.g., R1), and the random N-mer. At its other end, theportion of the sample nucleic acid can be linked to a region (e.g., R1c,or R1c partial) that is complementary or partially complementary to thesequencing primer binding site. A schematic representation of an examplesequence (in a linear configuration) is as follows:

-   P5-Barcode-R1-RandomNmer-Insert-R1c,partial    where “Insert” corresponds to the portion of the sample nucleic acid    copied during amplification. The sequencing primer binding site    (e.g., R1) and its partial complement (e.g., R1c, partial) at the    opposite end of the portion of the copied sample nucleic acid    (Insert) can intramolecularly hybridize to form a partial hairpin    structure as described elsewhere herein.

Following creation of the barcoded fragments of the sample nucleic acid,and as noted above, it may be desirable to further amplify thosefragments, as well as attach additional functional sequences to theamplified, barcoded fragments. This amplification may be carried outusing any suitable amplification process, including, e.g., PCR, LCR,linear amplification, or the like. Typically, this amplification may beinitiated using targeted primers that prime against the known terminalsequences in the created fragments, e.g., priming against one or both ofthe attachment sequence 3810, in FIGS. 38A-38F, and sequence 3814′.Further by incorporating additional functional sequences within theseprimers, e.g., additional attachment sequences such as P7, additionalsequencing primers, e.g., a read 2 or R2 priming sequence, as well asoptional sample indexing sequences, one can further configure theamplified barcoded fragments.

By way of example, following generation of partial hairpin amplicons,intramolecular hybridization of the partial hairpin amplicons can bedisrupted by contacting the partial hairpin amplicons with a primer thatis complementary to the duplex portion of the hairpin, e.g., sequence3814′, in order to disrupt the hairpin and prime extension along thehairpin structure. In many cases, it will be desirable to provide theseprimers with a stronger hybridization affinity than the hairpinstructure in order to preferentially disrupt that hairpin. As such, inat least one example, the primer comprises a locked nucleic acid (LNAs)or locked nucleic acid nucleotides. LNAs include nucleotides where theribonucleic acid base comprises a molecular bridge connecting the2′-oxygen and 4′-carbon of the nucleotide's ribose moiety. LNAsgenerally have higher melting temperatures and lower hybridizationenergies. Accordingly, LNAs can favorably compete with intramolecularhybridization of the partial hairpin amplicons by binding to any of thehybridized sequences of a partial hairpin amplicon. Subsequentamplification of the disrupted amplicons via primers comprising LNAs andother primers can generate linear products comprising any additionalsequences (including a sample index) to be added to the sequence.

For the example partial hairpinP5-Barcode-R1-RandomNmer-Insert-R1c,partial configuration describedabove, the partial hairpin can be contacted with a primer comprisingLNAs and a sequence complementary to R1c,partial (e.g., see FIG. 14C).The primer may also comprise the complement of any additional sequenceto be added to the construct. For example, the additional sequence(e.g., R2partial) may be a sequence that, when coupled to R1c,partial,generates an additional sequencing primer binding site (e.g., R2).Hybridization of the primer with the partial hairpin can disrupt thepartial hairpin's intramolecular hybridization and linearize theconstruct. Hybridization may occur, for example, such that the primerhybridizes with R1c,partial via its complementary sequence (e.g., seeFIG. 14C). Extension of the primer can generate a construct comprisingthe primer linked to a sequence complementary to the linearized partialhairpin amplicon. A schematic of an example construct is as follows:

-   P5c-Barcode,c-R1c-RandomNmer,c-Insert,c-R1,partial-R2partial,c    where P5c corresponds to the complement of P5, Barcode,c corresponds    to the complement of the barcode, RandomNmer,c corresponds to the    complement of the random N-mer, Insert,c corresponds to the    complement of the portion of the Insert, and R1,partial-R2partial,c    corresponds to the complement of R2.

Upon a further round of amplification with a second primer (e.g., P5,hybridizing at P5c), a linear construct comprising the partial hairpinamplicon sequence and a sequence complementary to the primer can begenerated. A schematic representation of an example configuration is asfollows:

-   P5-Barcode-R1-RandomNmer-Insert-R1c,partial-R2partial or-   P5-Barcode-R1-RandomNmer-Insert-R2    where the combined sequence of R1c,partial and R2partial can    correspond to an additional sequencing primer binding site (e.g.,    R2).

Additional sequences can be added to the construct using additionalrounds of such amplification, for however many additionalsequences/rounds of amplification are desired. For the exampleP5-Barcode-R1-RandomNmer-Insert-R2 construct described above, a primercomprising a sequence complementary to R2 (e.g., R2c), the complement ofa sample index sequence (e.g., S1c, SampleBarcode), and the complementof an additional sequencer primer binding site sequence (e.g., P7c) canbe hybridized to the construct at R2, via R2c of the primer (e.g., seeFIG. 14C). Extension of the primer can generate a construct comprisingthe primer linked to a sequence complementary to the construct. Aschematic representation of an example configuration is as follows:

-   P5c-Barcode,c-R1c-RandomNmer,c-Insert,c-R2,c-SIc-P7c

Upon a further round of amplification with a second primer (e.g., P5,hybridizing at P5c), a sequencer-ready construct comprising theconstruct sequence and a sequence complementary to the primer can begenerated. A schematic representation of an example configuration ofsuch a sequencer-ready construct is as follows:

P5-Barcode-R1-RandomNmer-Insert-R2-SampleIndex-PTAs an alternative, thestarting primer may comprise a barcode sequence, P7, and R2 (instead ofP5 and R1). A schematic representation of an example primer is asfollows:

-   P7-Barcode-R2-RandomNmer

Using an analogous amplification scheme as described above (e.g.,amplification with primers comprising LNAs, additional rounds ofamplification, etc.), an insert comprising a portion of a sample nucleicacid to be sequenced, P5, R1, and a sample index can be added to theprimer to generate a sequencer-ready product. A schematic representationof an example product is as follows:

-   P7-Barcode-R2-RandomNmer-Insert-R1-SampleIndex-P5

In other cases, a sample index may be added to a sample nucleic acidconcurrently with the addition of a barcode sequence to the samplenucleic acid. For example, a primer used to generate a barcoded samplenucleic acid may comprise both a barcode sequence and a sample index,such that when the barcode is coupled to the sample nucleic acid, thesample index is coupled simultaneously. The sample index may bepositioned anywhere in the primer sequence. In some cases, the primermay be a primer capable of generating barcoded sample nucleic acids viarandom amplification, such as PHASE amplification. Schematicrepresentations of examples of such primers include:

-   P5-Barcode-R1-SampleIndex-RandomNmer-   P5-Barcode-SampleIndex-R1-RandomNmer-   P5-SampleIndex-Barcode-R1-RandomNmer

Upon random priming of a sample nucleic acid with a respective primerand amplification of the sample nucleic acid in the partition, partialhairpin amplicons comprising a barcode sequence and a sample indexsequence can be generated. Schematic representations (shown in linearform) of examples of such partial hairpin amplicons generated from theabove primers include, respectively:

-   P5-Barcode-R1-SampleIndex-RandomNmer-Insert-R1c,partial-   P5-Barcode-SampleIndex-R1-RandomNmer-Insert-R1c,partial-   P5-SampleIndex-Barcode-R1-RandomNmer-Insert-R1c,partial    R1c, partial can intramolecularly hybridize with its complementary    sequence in R1 to form a partial hairpin amplicon.

By way of example, in some cases, following the generation of partialhairpin amplicons, additional sequences (e.g., functional sequences likeR2 and P7 sequences) can be added to the partial hairpin amplicons, suchas, for example, in bulk. In analogous fashion to amplification methodsdescribed elsewhere herein, primers that include these additionalfunctional sequences may be used to prime the replication of the partialhairpin molecule, e.g., by priming against the 5′ end of the partialhairpin, e.g., the R1c sequence, described above. In many cases, it willbe desirable to provide a higher affinity primer sequence, e.g., tooutcompete rehybridization of the hairpin structure, in order to providegreater priming and replication. In such cases, tighter binding primersequences, e.g., that include in their sequence one or more higheraffinity nucleotide analogues, like LNAs or the like, may be used todisrupt partial hairpin amplicons and add additional sequences to theamplicons. For example, with reference to the example described above, aprimer may comprise LNAs, a sequence complementary to R1c,partial and asequence comprising the complement to R2partial, such that when theprimer is extended and the resulting product further amplified via a P5primer, R1c,partial and R2partial are joined to generate R2. Schematicrepresentations of examples of such constructs generated from the aboveprimers include, respectively:

-   P5-Barcode-R1-SampleIndex-RandomNmer-Insert-R2-   P5-Barcode-SampleIndex-R1-RandomNmer-Insert-R2-   P5-SampleIndex-Barcode-R1-RandomNmer-Insert-R2

As noted above, additional rounds of amplification cycles may be used toadd additional sequences to the constructs. For example, a primer maycomprise a sequence complementary to R2 and a sequence comprising thecomplement to P7, such that when the primer is extended and theresulting product further amplified via a P5 primer, P7 is linked to R2and a sequencer-ready construct is generated. Schematic representationsof examples of such sequencer-ready constructs generated from the aboveprimers include, respectively:

-   P5-Barcode-R1-SampleIndex-RandomNmer-Insert-R2-P7-   P5-Barcode-SampleIndex-R1-RandomNmer-Insert-R2-P7-   P5-SampleIndex-Barcode-R1-RandomNmer-Insert-R2-P7

Combining a barcode and a sample index into a primer capable ofamplifying regions of a sample nucleic acid (e.g., via PHASEamplification) may enable parallelization of sample indexing. Sets ofprimers may be used to index nucleic acids from different samples. Eachset of primers may be associated with nucleic acid molecules obtainedfrom a particular sample and comprise primers comprising a diversity ofbarcode sequences and a common sample index sequence.

In some cases, it may be desirable to attach additional sequencesegments to the 5′ end of the partial hairpin molecules describedherein, not only to provide additional functionality to the amplifiedfragment of the sample nucleic acid as described above, but also toensure more efficient subsequent processing, e.g., amplification and/orsequencing, of those molecules. For example, where a partial hairpinmolecule is subjected to extension reaction conditions, it may besusceptible to filling in of the partial hairpin structure, by primingits own ‘filling in’ reaction through extension at the 5′ terminus. As aresult, a complete hairpin structure may be created that is moredifficult to amplify, by virtue of the greater stability of its duplexportion. In such cases, it may be desirable to preferentially attachadditional sequence segment(s) that is not complementary to the opposingend sequence, in order to prevent the formation of a complete hairpinstructure. In one exemplary process, the LNA primers described above forthe amplification of the partial hairpin structures, may be providedwith additional overhanging sequence, including, e.g., the R2complementary sequence described above, as well as potentiallycomplementary sequences to other functional sequence components, e.g.,attachment sequences like P7, sample index sequences, and the like.Subjecting the partial hairpin and primer to the extension reactiondescribed above for amplification of that partial hairpin, will alsoresult in extension of the partial hairpin along the overhangingsequence on the LNA primer. The extended sequence may comprise simply anon-complementary sequence, or it may comprise additional functionalsequences, or their complements as noted above, such that the extensionreaction results in attachment of those functional sequences to the 5′terminus of the partial hairpin structure.

In alternative aspects, additional sequence segments may be ligated tothe 5′ end of the partial hairpin structure where such sequence segmentsare not complementary to the non-overlapped portion of the hairpinstructure. The foregoing are schematically illustrated in FIG. 40. Asshown in path A, a partial hairpin structure, when subjected to primerextension conditions, may act as its own primer and have its 5′ sequenceextended, as shown by the dashed arrow, until it forms a complete ornearly complete hairpin structure, e.g., with little or no overhangsequence. This full hairpin structure will possess far greater duplexstability, thereby potentially negatively impacting the ability todisrupt the hairpin structure to prime its replication, even whenemploying higher affinity primers, e.g., LNA containing primers/probes.

In order to minimize this possibility, as shown in both paths B and C, aseparate sequence segment 4006 is added to the 5′end of the hairpinstructure, to provide a partial hairpin with non-complementary tailsequences 4008, in order to prevent the generation of the complete ornearly complete hairpin structure. As shown, this may be accomplished ina number of different ways. For example, in a first process shown inpath B, an invading probe 4010 may be used to disrupt the partialhairpin structure and hybridize to sequence segment 4012. Such invadingprobes may be provided with higher affinity binding than the inherentpartial hairpin structure, e.g., through use of higher affinitynucleotide analogues such as LNAs or the like. In particular, thatportion of the invader sequence 4010 that hybridizes to sequence segment4012 may comprise LNAs within its sequence in the same fashion describedherein for use with LNA primer sequences used in subsequentamplification.

Extension of the 5′ portion of the partial hairpin (and sequence segment4012) as shown by the dashed arrow in path B, then appends the sequence4006 to the 5′ terminus of the partial hairpin structure to providestructure 4008. Alternatively, sequence 4006 may be ligated to the 5′end of the partial hairpin structure 4002 (or sequence segment 4012). Asshown in path C, this achieved through the use of a splint sequence 4014that is partially complementary to sequence 4006 and partiallycomplementary to sequence 4012, in order to hold sequence 4006 adjacentto sequence segment 4012 for ligation. As will be appreciated, thesplint sequence 4014 may again utilize a higher affinity invading probe,like probe 4010, to disrupt the hairpin structure and hybridize tosequence segment 4012. In particular, again, that portion of splintsequence 4014 that is intended to hybridize to sequence segment 4012 maybe provided with one or more LNA nucleotide analogues within itssequence, in order to preferentially disrupt the partial hairpinstructure 4002, and allow ligation of sequence 4006 to its 5′ end.

In some cases, a microfluidic device (e.g., a microfluidic chip) may beuseful in parallelizing sample indexing. Such a device may compriseparallel modules each capable of adding a barcode sequence and a sampleindex to nucleic acid molecules of a sample via primers comprising boththe barcode sequence and the sample index. Each parallel module maycomprise a primer set comprising a different sample index, such that thesample processed in each module is associated with a different sampleindex and set of barcodes. For example, a microfluidic device with 8modules may be capable of sample indexing 8 different samples. Followingbarcoding and sample indexing via attachment of the sequences to asample nucleic acid, bulk addition of additional sequences (e.g., R2,P7, other barcode sequences) via, for example, serial amplification canbe used to generate sequencer-ready products as described elsewhereherein.

In some cases, sample indexing may be achieved during barcoding withoutthe inclusion of a separate sample index sequence in a primer used toattach a barcode to a sample nucleic acid. In such cases, a barcodesequence, for example, may also serve as a sample index. An exampleconfiguration of a sequencer-ready construct with a sequence functioningas both a barcode sequence and a sample index is as follows:

-   P5-BSI-R1-RandomNmer-Insert-R2-P7    where “BSI” is the sequence functioning as both a barcode sequence    and a sample index.    A sequencer-ready product may comprise a barcode sequence that can    be used to align sequence reads and provide a sequence for a sample    nucleic acid. The sequencer-ready product may be generated, for    example, using PHASE amplification and subsequent bulk amplification    as described elsewhere herein. Moreover, the barcode sequence may    belong to a particular set of known barcode sequences. The set of    barcode sequences may be associated with a particular sample, such    that identification of the sample from which a particular sequencing    read originates can be achieved via the read barcode sequence. Each    sample can be associated with a set of known barcode sequences, with    each barcode sequence set comprising barcode sequences that do not    overlap with barcode sequence in other barcode sets associated with    other samples. Thus, the uniqueness of a barcode sequence and its    uniqueness amongst different sets of barcode sequences may be used    for multiplexing.

For example, a sequencing read may comprise the barcode sequence“GAGCCG”. Barcode sequence “GAGCCG” may be a barcode sequence in a setof known barcode sequences associated with Sample A. The sequence is notfound in a set of known barcode sequences associated with anothersample. Upon reading the sequence “GAGCCG”, it can be determined thatthe sequence read is associated with Sample A because the sequence“GAGCCG” is unique to the set of barcode sequences associated withSample A. Moreover, another sequencing read may comprise the barcodesequence “AGCAGA”. Barcode sequence “AGCAGA” may be a barcode sequencein a set of known barcode sequences associated with Sample B. Thesequence is not found in a set of known barcode sequences associatedwith another sample. Upon reading the sequence “AGCAGA”, it can bedetermined that the sequence read is associated with Sample B because“AGCAGA” is unique to the set of barcode sequences associated withSample B.

In another example, a sample index sequence may be embedded in a randomsequence of a primer used in one or more amplification reactions toattach a barcode to a sample nucleic acid. For example, a primer maycomprise a barcode sequence and a random sequence that can be used torandomly prime a sample nucleic acid and attach the barcode sequence tothe sample nucleic acid. In some cases, the random sequence may be apseudo-random sequence such that particular bases of the random sequenceare conserved between all primers. The pattern of the conserved basesmay be used as a sample index, such that all sequencer-ready productsobtained from a particular sample all comprise the conserved pattern ofbases in the random sequence region. Each sample can be associated witha different pattern of conserved bases and, thus, multiplexing can beachieved. In some cases, the pattern is a contiguous sequence region ofa pseudo-random sequence (e.g., “NNNATACNNN” (SEQ ID NO: 1)) or in othercases, the pattern is a non-contiguous sequence region of apseudo-random sequence (e.g., “NCNGNNAANN” (SEQ ID NO: 2)), where “N”corresponds to a random base. Moreover, any suitable number of bases maybe conserved in a pseudo-random sequence in any pattern and the examplesdescribed herein are not meant to be limiting. An example configurationof a sequencer-ready construct with a sequence functioning as both abarcode sequence and a sample index is as follows:

-   P5-Barcode-R1-NQNQNNQQNN-Insert-R2-P7    where “Q” is a conserved base in the random region

For example, a sequencer-ready product may comprise a 10-merpseudo-random sequence “NCNGNNAANN” (SEQ ID NO: 2), where the secondbase (“C”), fourth base (“G”), seventh base (“A”), and eighth base (“A”)of the pseudo-random sequence are conserved for all sequencer-readyproducts generated from Sample A. A sequencing read may comprise such apattern of conserved bases in the random sequence region. Upon readingthe conserved base pattern, it can be determined that the sequence readis associated with Sample A because the “NCNGNNAANN” (SEQ ID NO: 2)conserved pattern of bases is associated with Sample A. Moreover, asequencer-ready product may comprise a 10-mer pseudo-random sequence“NNGCNGNGNN” (SEQ ID NO: 3), where the third base (“G”), fourth base(“C”), sixth base (“G”), and eighth base (“G”) of the pseudo-randomsequence are conserved for all sequencer-ready products generated fromSample B. A sequencing read may comprise such a pattern of conservedbases in the random sequence region. Upon reading the conserved basepattern, it can be determined that the sequence read is associated withSample B because the “NNGCNGNGNN” (SEQ ID NO: 3) conserved pattern ofbases is associated with Sample B.

In other cases, a sample index may be added to a sample nucleic acidprior to the addition of a barcode sequence to the sample nucleic acid.For example, a sample nucleic acid may be pre-amplified in bulk suchthat resulting amplicons are attached to a sample index sequence priorto barcoding. For example, sample may be amplified with a primercomprising a sample index sequence such that the sample index sequencecan be attached to the sample nucleic acid. In some cases, the primermay be a random primer (e.g., comprising a random N-mer) andamplification may be random. Produced amplicons that comprise the sampleindex can then be barcoded using any suitable method, includingbarcoding methods described herein.

Sample nucleic acid molecules can be combined into partitions (e.g.,droplets of an emulsion) with the primers described above. In somecases, each partition can comprise a plurality of sample nucleic acidmolecules (e.g., smaller pieces of a larger nucleic acid). In somecases, no more than one copy of a unique sample nucleic acid molecule ispresent per partition. In some cases, each partition can generallycomprise primers comprising an identical barcode sequence and a samplepriming sequence (e.g., a variable random-Nmer, a targeted N-mer), withthe barcode sequence generally differing between partitions. In suchcases, each partition (and, thus, sample nucleic acid in the partition)can be associated with a unique barcode sequence and the unique barcodesequence can be used to determine a sequence for the barcoded samplenucleic acid generated in the partition.

In some cases, upon generation of barcoded sample nucleic acids, thebarcoded sample nucleic acids can be released from their individualpartitions, pooled, and subject to bulk amplification schemes to addadditional sequences (e.g., additional sequencing primer binding sites,additional sequencer primer binding sites, additional barcode sequences,sample index sequences) common to all downstream sequencer-readyproducts. In cases where the partitions are droplets of an emulsion, theemulsion may be broken and the barcoded sample nucleic acids pooled. Asample index can be added in bulk to the released, barcoded samplenucleic acids, for example, using the serial amplification methodsdescribed herein. Where a sample index is added in bulk, eachsequencer-ready product generated from the same sample will comprise thesame sample index that can be used to identify the sample from which theread for the sequencer-ready product was generated. Where a sample indexis added during barcoding, each primer used for barcoding may comprisean identical sample index sequence, such that each sequencer-readyproduct generated from the same sample will comprise the same sampleindex sequence.

Partitioning of sample nucleic acids to generate barcoded (or barcodedand sample indexed) sample nucleic acids and subsequent addition ofadditional sequences (e.g., including a sample index) to the barcodedsample nucleic acids can be repeated for each sample, using a differentsample index for each sample. In some cases, a microfluidic dropletgenerator may be used to partition sample nucleic acids. In some cases,a microfluidic chip may comprise multiple droplet generators, such thata different sample can be processed at each droplet generator,permitting parallel sample indexing. Via each different sample index,multiplexing during sequencing can be achieved.

Upon the generation of sequencer-ready oligonucleotides, thesequencer-ready oligonucleotides can then be provided to a sequencingdevice for sequencing. Thus, for example, the entire sequence providedto the sequencing device may comprise one or more adaptors compatiblewith the sequencing device (e.g. P5, P7), one or more barcode sequences,one or more primer binding sites (e.g. Read1 (R1) sequence primer, Read2(R2) sequencing primer, Index primer), an N-mer sequence, a universalsequence, the sequence of interest, and combinations thereof. Thebarcode sequence may be located at either end of the sequence. In somecases, the barcode sequence may be located between P5 and Read1 sequenceprimer binding site. In other cases, the barcode sequence may be locatedbetween P7 and Read 2 sequence primer binding site. In some cases, asecond barcode sequence may be located between P7 and Read 2 sequenceprimer binding site. The index sequence primer binding site may beutilized in the sequencing device to determine the barcode sequence.

The configuration of the various components (e.g., adaptors, barcodesequences, sample index sequences, sample sequence, primer bindingsites, etc.) of a sequence to be provided to a sequencer device may varydepending on, for example the particular configuration desired and/orthe order in which the various components of the sequence is added. Anysuitable configuration for sequencing may be used and any sequences canbe added to oligonucleotides in any suitable order. Additional sequencesmay be added to a sample nucleic acid prior to, during, and afterbarcoding of the sample nucleic acid. For example, a P5 sequence can beadded to a sample nucleic acid during barcoding and P7 can be added inbulk amplification following barcoding of the sample nucleic acid.Alternatively, a P7 sequence can be added to a sample nucleic acidduring barcoding and a P5 sequence can be added in bulk amplificationfollowing barcoding of the sample nucleic acid. Example configurationsdisplayed as examples herein are not intended to be limiting. Moreover,the addition of sequence components to an oligonucleotide viaamplification is also not meant to be limiting. Other methods, such as,for example, ligation may also be used. Furthermore, adaptors, barcodesequences, sample index sequences, primer binding sites, sequencer-readyproducts, etc. described herein are not meant to be limiting. Any typeof oligonucleotide described herein, including sequencer-ready products,may be generated for any suitable type of sequencing platform (e.g.,Illumina sequencing, Life Technologies Ion Torrent, Pacific BiosciencesSMRT, Roche 454 sequencing, Life Technologies SOLiD sequencing, etc.)using methods described herein.

Sequencer-ready oligonucleotides can be generated with any adaptorsequence suitable for a particular sequencing platform using methodsdescribed herein. For example, sequencer-ready oligonucleotidescomprising one or more barcode sequences and P1 and A adaptor sequencesuseful in Life Technologies Ion Torrent sequencing may be generatedusing methods described herein. In one example, beads (e.g., gel beads)comprising an acrydite moiety linked to a P1 sequence via a disulfidebond may be generated. A barcode construct may be generated thatcomprises a P1sequence, a barcode sequence, and a random N-mer sequence.The barcode construct may enter an amplification reaction (e.g., in apartition, such as a fluidic droplet) to barcode sample nucleic acid.Barcoded amplicons may then be subject to further amplification in bulkto add the A sequence and any other sequence desired, such as a sampleindex. Alternatively, P1 and A sequences can be interchanged such that Ais added during sample barcoding and P1 is added in bulk. The completesequence can then be entered into an Ion Torrent sequencer. Otheradaptor sequences (e.g., P1 adaptor sequence for Life Technologies SOLiDsequencing, A and B adaptor sequences for Roche 454, etc.) for othersequencing platforms can be added in analogous fashion.

Although described herein as generating partial hairpin molecules, andin some cases, preventing formation of complete hairpins, in some cases,it may be desirable to provide complete hairpin fragments that includethe barcode sequences described herein. In particular, such completehairpin molecules may be further subjected to conventional samplepreparation steps by treating the 3′ and 5′ end of the single hairpinmolecule as one end of a double stranded duplex molecule in aconventional sequencing workflow. In particular, using conventionalligation steps, one could readily attach the appropriate adaptersequences to both the 3′ and 5′ end of the hairpin molecule in the samefashion as those are attached to the 3′ and 5′ termini of a duplexmolecule. For example, in case of an Illumina based sequencing process,one could attach a standard Y adapter that includes the P5 and P7adapters and R1 and R2 primer sequences, to one end of the hairpin as ifit were one end of a duplex molecule, using standard Illumina protocols.

Methods for Reducing Undesired Amplification Products (Partial HairpinAmplification for Sequencing (PHASE))

A random N-mer sequence may be used to randomly prime a sample, such asgenomic DNA (gDNA). In some embodiments, the random N-mer may comprise aprimer. In some cases, the random N-mer may prime a sample. In somecases, the random N-mer may prime genomic DNA. In some cases, the randomN-mer may prime DNA fragments.

Additionally, a random N-mer sequence may also be attached to anotheroligonucleotide. This oligonucleotide may be a universal sequence and/ormay contain one or more primer read sequences that may be compatiblewith a sequencing device (e.g. Read 1 primer site, Read 2 primer site,Index primer site), one or more barcode sequences, and one or moreadaptor segments that may be compatible with a sequencing device (e.g.P5, P7). Alternatively, the oligonucleotide may comprise none of theseand may include another sequence.

Via subsequent amplification methods, priming of a sample nucleic acidwith a random N-mer may be used to attach an oligonucleotide sequence(e.g., an oligonucleotide sequence comprising a barcode sequence) linkedto a random N-mer to the sample nucleic acid, including a sample nucleicacid to be sequenced. Utilizing random primers to prime a sample mayintroduce significant sequence read errors, due to, for example, theproduction of undesired amplification products.

To mitigate undesired amplification products, at least a subsection ofan oligonucleotide sequence may be substituted with dUTPs or uracilcontaining nucleotides in place of dTTPs or thymine containingnucleotides, respectively. In some cases, substitution may be complete(e.g., all thymine containing nucleotides are substituted with uracilcontaining nucleotides), or may be partial such that a portion of anoligonucleotide's thymine containing nucleotides are substituted withuracil containing nucleotides. In some cases, thymine containingnucleotides in all but the last about 10 to about 20, last about 10 to30, last about 10 to 40, or last about 5 to 40 nucleotides of anoligonucleotide sequence adjacent to a random N-mer sequence aresubstituted with dUTPs or uracil containing nucleotides. In addition, apolymerase that does not accept or process uracil-containing templatesmay be used for amplification of the sample nucleic acid. In this case,the non-uracil containing portion of about 10 to about 20 nucleotidesmay be amplified and the remaining portion containing the dUTPs oruracil containing nucleotides may not be amplified. In some cases, theportion of an oligonucleotide sequence comprising dUTPs or uracilcontaining nucleotides may be adjacent to the N-mer sequence. In somecases, the portion of an oligonucleotide sequence comprising dUTPs oruracil containing nucleotides may be adjacent to the barcode sequence.Any portion of an oligonucleotide sequence, including an adaptorsegment, barcode, or read primer sequence may comprise dUTPs or uracilcontaining nucleotides (e.g., substituted for thymine containingnucleotides), depending upon the configuration of the oligonucleotidesequence.

Moreover, the number and positioning of uracil containingnucleotide-for-thymine containing nucleotide substitutions in anoligonucleotide may be used, for example, to tune the size of partialhairpin products obtained with amplification methods described belowand/or to tune the binding of the polymerase enzyme with a uracilcontaining primer sequence. Additionally, free uracil containingnucleotides, e.g., UTP or an analogue thereof, may also be providedwithin the reaction mixture, e.g., within the partition, at a desiredconcentration to mediate polymerase/uracil-primer binding kinetics. Insome cases, smaller partial hairpin products may give rise to moreaccurate sequencing results. Accordingly, an oligonucleotide maycomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more uracilcontaining nucleotide-for-thymine containing nucleotide substitutionsdepending upon, for example, the desired length of partial hairpinproducts generated from the oligonucleotide.

Upon random priming of a sample nucleic acid with a random N-mer linkedto an oligonucleotide sequence (e.g., an oligonucleotide sequencecomprising uracil containing nucleotides described above) FIG. 15A, afirst round of amplification (e.g., using a polymerase that does notaccept or process a uracil containing nucleotide as a template) mayresult in the attachment of the oligonucleotide sequence to a complementof the sample nucleic acid, FIG. 15B and FIG. 15C. Upon priming (via therandom N-mer) and further amplification of the amplification productwith another copy of the oligonucleotide sequence comprising the randomN-mer (FIG. 15D), an amplification product comprising theoligonucleotide sequence, a portion of the sample nucleic acid sequence,and a partial complementary oligonucleotide sequence (e.g.,complementary to the portion of the oligonucleotide sequence notcomprising uracil containing nucleotides) at an end of the amplificationproduct opposite the oligonucleotide sequence, can be generated. Thepartial complementary oligonucleotide sequence and the oligonucleotidesequence can hybridize to form a partial hairpin that, in some cases,can no longer participate in nucleic acid amplification. A partialhairpin can be generated because a portion of the originaloligonucleotide sequence comprising uracil containing nucleotides wasnot copied. Amplification can continue for a desired number of cycles(e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles), up until alloligonucleotide sequences comprising random N-mers have been exhausted(FIGS. 15E-15G).

In some embodiments, to ensure priming of sample nucleic acid (e.g.,genomic DNA (gDNA)) with only a random N-mer and not portions of anattached oligonucleotide sequence, the oligonucleotide sequence may beblocked via hybridization of a blocker oligonucleotide (e.g., blackdumbbell in FIGS. 15A-15G). A blocker oligonucleotide (also referred toas an oligonucleotide blocker elsewhere herein) may be hybridized to anyportion of an oligonucleotide sequence, including a barcode sequence,read primer site sequence, all or a portion of a uracil containingportion of the oligonucleotides, or all or any other portion of theoligonucleotides, or other sequence therein. A blocker oligonucleotidemay be DNA or RNA. In some cases, a blocker oligonucleotide may compriseuracil containing nucleotide-for-thymine containing nucleotidesubstitutions. In some cases, all of the thymine containing nucleotidesof a blocker oligonucleotide may be substituted with uracil containingnucleotides. In some cases, a portion of the thymine containingnucleotides of a blocker oligonucleotide may be substituted with uracilcontaining nucleotides. In some cases, a blocker oligonucleotide maycomprise locked nucleic acid (LNA), an LNA nucleotide, bridged nucleicacid (BNA), and/or a BNA nucleotide. Moreover a blocker oligonucleotidemay be of any suitable length necessary for blocker functionality. Ablocker oligonucleotide may be of length suitable to block a portion ofan oligonucleotide or may be of the same or of substantially the samelength of an oligonucleotide it is designed to block. The blockeroligonucleotide may ensure that only random N-mers bind to the samplenucleic acid (e.g., genomic DNA) and not other portions of theoligonucleotide sequence.

The stoichiometric ratio of a blocker oligonucleotide to oligonucleotide(e.g., blocker oligonucleotide:oligonucleotide) may vary. For example,the blocker oligonucleotide:oligonucleotide stoichiometric ratio may beabout 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50,0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10,1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70,1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60,2.70, 2.80, 2.90, 3.00, 3.50, 4.00, 4.50, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 10.0, 20, 30, 40, 50, 100 or more. In some cases, theblocker oligonucleotide:oligonucleotide stoichiometric ratio may be atleast about 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45,0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05,1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65,1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.10, 2.20, 2.30, 2.40, 2.50,2.60, 2.70, 2.80, 2.90, 3.00, 3.50, 4.00, 4.50, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 10.0, 20, 30, 40, 50, 100 or more. In some cases,the blocker oligonucleotide:oligonucleotide stoichiometric ratio may beat most about 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00,1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60,1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.10, 2.20, 2.30, 2.40,2.50, 2.60, 2.70, 2.80, 2.90, 3.00, 3.50, 4.00, 4.50, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 20, 30, 40, 50, or 100.

Moreover, incorporation of a blocker moiety (e.g., via adideoxynucleotide (ddNTP), ddCTP, 0, ddGTP, ddTTP, etc. at the 3′ or 5′end of the blocker oligonucleotide) to a blocker oligonucleotide and/orthe inclusion of uracil containing nucleotides (e.g., substituted forall or a portion of thymine containing nucleotides) in a blockeroligonucleotide may prevent preferential binding of blocked portions ofthe blocked oligonucleotide sequence to the sample nucleic acid.Additional examples of blocker moieties include 3′ phosphate, a blocked3′ end, 3′ddCTP, C3 Spacer (/3SpC3/), Dideoxy-C(/3ddC/). Blockeroligonucleotides may be cleaved from an oligonucleotide sequence byRNAse, RNAseH, an antisense DNA oligonucleotide, and/or alkalinephosphatase.

In some cases, an oligonucleotide sequence may be blocked with a blockeroligonucleotide such that the oligonucleotide sequence comprises ablocked 5′ end, comprises a blocked 3′ end, may be entirely blocked(e.g., may be entirely blocked, except for its random N-mer sequence),or may be blocked at another location (e.g., a partial sequence of theoligonucleotide, different from an oligonucleotide sequence's randomN-mer). In some cases, an oligonucleotide sequence may comprise aplurality of blockers, such that multiple sites of the oligonucleotideare blocked. In some cases, an oligonucleotide sequence may compriseboth a blocked 3′ end and uracil containing nucleotides. In some cases,an oligonucleotide sequence comprising uracil containing nucleotides anda blocked 3′ end may be adjacent to the N-mer sequence. In some cases,an oligonucleotide sequence may comprise a blocked 3′ end. In somecases, an oligonucleotide sequence may comprise uracil containingnucleotides. In some cases, an oligonucleotide sequence may compriseboth a blocked 5′ end and uracil containing nucleotides.

In some cases, the oligonucleotide sequence comprising uracil containingnucleotides and a blocked 3′ end may be adjacent to the N-mer sequence.In some cases, the oligonucleotide sequence comprising uracil containingnucleotides and a blocked 3′ end may be adjacent to the barcodesequence. In some cases, the oligonucleotide sequence may comprise ablocked 3′ end. In some cases, the oligonucleotide sequence may compriseuracil containing nucleotides. In some cases, the oligonucleotidesequence may comprise both the blocked 3′ end and uracil containingnucleotides. Addition of a blocker oligonucleotide may preventpreferential binding to portions of the universal sequence, which maynot be desired to be amplified.

In some cases, an oligonucleotide suitable for priming a sample nucleicacid via its random N-mer may also comprise a blocking sequence that canfunction in the same role as a blocker oligonucleotide. For example, anoligonucleotide may be arranged in a hairpin configuration with ablocking sequence that can function in the same role as a blockeroligonucleotide. An example oligonucleotide comprising a random N-mer,an R1c sequence, a P5 sequence, a barcode sequence, and an R1 sequencemay be configured as follows:

-   5′-RandomNmer-R1c-P5-Barcode-R1-3′

The R1 sequence and R1c sequence of the oligonucleotide may hybridize togenerate a hairpin with a hairpin loop comprising the P5 and Barcodesequences. The R1c sequence can function in the same role as a blockeroligonucleotide such that priming of sample nucleic acid with theoligonucleotide occurs via only the oligonucleotide's random N-mer. Insome cases, one or more cleavage sites (e.g., a restriction site, acleavage site, an abasic site, etc.) may be included in anoligonucleotide arranged as a hairpin with a blocking sequence,including an oligonucleotide's hairpin loop, to separate sequencecomponents of the oligonucleotide downstream, if desired. Separation mayoccur, for example, via an enzymatic reaction, oxidation-reduction,radiation (e.g., UV-light), the addition of heat, or other suitablemeans.

An example uracil containing nucleotide-substituted oligonucleotidesequence linked to a random N-mer is depicted in FIG. 14B. Specifically,a random primer (e.g., a random N-mer), of about 8N-12N in length, 1404,may be linked with an oligonucleotide sequence. The random N-mer may beused to randomly prime and extend from a sample nucleic acid, such as,genomic DNA (gDNA). The oligonucleotide sequence comprises: (1)sequences for compatibility with a sequencing device, such as, a flowcell (e.g. Illumina's P5, 1401, and Read 1 Primer sites, 1402) and (2) abarcode (BC), 1403, (e.g., 6-12 base sequences). Furthermore, the Read 1Primer site 1402 of the oligonucleotide sequence may be hybridized witha blocking oligonucleotide comprising uracil containing nucleotides anda blocker moiety at its 3′ end (e.g. 3′ddCTP, indicated by an “X”). Theblocking oligonucleotide can be used to promote priming of a samplenucleic acid with only the random N-mer sequence and preventpreferential binding of the oligonucleotide sequence to portions of thesample nucleic acid that are complementary to the Read 1 Primer site,1402. Optionally, to further limit product lengths, a small percentageof terminating nucleotides (e.g., 0.1-2% acyclonucleotides (acyNTPs))(FIG. 16B) may be included in oligonucleotide sequences to reduceundesired amplification products.

An example of partial hairpin amplification for attaching a uracilcontaining nucleotide-substituted oligonucleotide sequence comprising arandom N-mer to a sample nucleic acid (e.g., genomic DNA (gDNA)) isdepicted in FIGS. 15A-15G. First, initial denaturation of the samplenucleic acid may be achieved at a denaturation temperature (e.g., 98°C., for 2 minutes) followed by priming of a random portion of the samplenucleic acid with the random N-mer sequence at a priming temperature(e.g., 30 seconds at 4° C.), FIG. 15A. The oligonucleotide sequence ishybridized with a blocking oligonucleotide (black dumbbell in FIGS.15A-15G), to ensure that only the random N-mer primes the sample nucleicacid and not another portion of the oligonucleotide sequence.Subsequently, sequence extension (e.g., via polymerase that does notaccept or process a uracil containing nucleotide as a template) mayfollow as the temperature ramps to higher temperature (e.g., at 0.1°C./second to 45° C. (held for 1 second)) (FIG. 15A). Extension may thencontinue at elevated temperatures (e.g., 20 seconds at 70° C.),continuing to displace upstream strands and create a first phase ofredundancy (FIG. 15B). Denaturation of the amplification product maythen occur at a denaturing temperature (e.g., 98° C. for 30 seconds) torelease the sample nucleic acid and amplification product for additionalpriming.

After the first cycle, amplification products have a single 5′ tag (FIG.15C) comprising the oligonucleotide sequence. These aforementioned stepsare repeated to prime the amplification product and sample nucleic acidwith the oligonucleotide sequence via its random N-mer. The blacksequence indicates portions of the added 5′ tags (added in cycle 1) thatcomprise uracil containing nucleotides and thus, will not be copied uponpriming and amplification of the amplification product (FIG. 15D).Following a second round of amplification, both 5′ tagged products and3′ & 5′ tagged products may be generated (FIG. 15E). The 3′ & 5′ taggedproducts comprise a full oligonucleotide sequence at one end, the samplenucleic acid sequence, and a sequence partially complementary to theoligonucleotide sequence (e.g., complementary to regions of theoligonucleotide sequence not comprising uracil containing nucleotides)at the other end of the oligonucleotide. The oligonucleotide sequencemay hybridize with its partially complementary sequence to generate apartial hairpin structure (FIG. 15F. Amplification can continuerepeatedly for a desired number of cycles (e.g., up to 20 times), upuntil all oligonucleotide sequences have been exhausted (FIG. 15G).

Partial hairpin formation may prevent generating a copy of a copy andmay instead encourage only copies of the original template to beproduced, thus reducing potential amplification bias, and otherartifacts. Partial hairpin formation may encourage segregation of thedesired product and may reduce production of copies.

Desirable properties for the uracil-non-reading polymerase to form thepartial hairpin may include an exonuclease deficient polymerase (e.g.,having low exonuclease activity, having substantially no exonucleaseactivity, having no exonuclease activity), strand displacingcapabilities (e.g., a thermostable strand displacing polymerase enzyme),residual activity at temperatures <50° C., and discrimination againsturacil containing nucleotides v thymine containing nucleotides. Examplesof such polymerases may include 9 degrees North, modified (NEB), exominus Pfu, Deep Vent exo minus, Vent exo minus, and homologs thereof.Moreover, a polymerase with low exonuclease activity may be a polymerasewith less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10%,less than 5%, or 0% exonuclease activity of a thermally stablepolymerase with normal exonuclease activity (e.g., Taq polymerase). Insome cases, a polymerase used for partial hairpin amplification may becapable of strand-displacement. In some cases, limiting the length ofthe amplified sequence may reduce undesired amplification products,wherein longer length products may include undesired upstream portionssuch as a barcode sequence. The amplified product length may be limitedby inclusion of terminating nucleotides. An example of a terminatingnucleotide may include an acyclonucleotide (acyNTPs). Terminatingnucleotides may be present at about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% of the amplified productlength. In some cases, terminating nucleotides may be present at morethan about 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%,2.3%, 2.4%, 2.5%, or more of the amplified product length. In somecases, terminating nucleotides may be present at less than about 0%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, or2.5% of the amplified product length.

Amplification product length may also be controlled by pre-amplificationof sample nucleic acid prior to initiation of PHASE amplification. Forexample, a random N-mer may be used for pre-amplification of the samplenucleic acid. A random N-mer may be used to prime a sample nucleic acidfollowed by extension of the primer using suitable thermal cyclingconditions. Product length can be controlled by thermal cyclingconditions (e.g., number of thermal cycles, temperatures utilized, cycletime, total run time, etc.) in addition to the random priming of thesample nucleic acid. In some cases, pre-amplification products smallerthan the original sample nucleic acid can be obtained. Amplificationproducts generated during pre-amplification may then be entered into aPHASE amplification and barcoded as described above.

As shown in FIG. 17, addition of a blocking oligonucleotide may reducestart site bias by 50%. Incorporation of uracil containing nucleotidesinstead of thymine containing nucleotides into the universal sequenceand using a polymerase that does not accept or process uracil-containingtemplates, may significantly reduce sequencing errors, as reported inFIG. 21 and FIG. 22. For example, Q40 error may be reduced from about0.002 to about 0.001, unmapped fraction ends may be reduced from about0.996 to about 0.03, median insert size may be reduced from about 399 toabout 310, IQR insert size may be reduced from about 413 to about 209,and zero coverage fraction may be reduced from about 0.9242 to about0.0093.

Amplification schemes that do not involve the substitution of thyminecontaining nucleotides with uracil containing nucleotides are alsoenvisioned for generating partial hairpin species. In some cases, otherspecies unable to be recognized or be copied by a polymerase (e.g.,methylated bases, abasic sites, bases linked to bulky side groups, etc.)may be used in place of uracil containing nucleotides to generatepartial hairpin amplicons. In some cases, full hairpin amplicons may begenerated and processed post-synthesis to generate partial hairpinspecies. In some cases, full hairpin amplicons may be generated andportions subsequently removed to generate partial hairpin species. Forexample, as shown in FIG. 34A, full hairpin amplicons 3401 can begenerated via the amplification scheme depicted in FIGS. 15A-15G whenoligonucleotide primers comprising random N-mers do not comprise uracilcontaining nucleotides and/or a polymerase capable of accepting orprocessing a uracil containing template is used for amplification. Upongeneration of the full hairpin amplicons 3401, the full hairpinamplicons can be enzymatically (e.g., via a restriction enzyme or othersite specific enzyme such as a nickase) or chemically nicked 3403 at oneor more appropriate sites to generate partial hairpin species 3402.

In some cases, full hairpin amplicons may be generated and portionsadded to the full hairpin amplicons to generate partial hairpin species.For example, a primer comprising a sequencing primer binding site (e.g.,R1) coupled to a random N-mer and not comprising uracil containingnucleotides may be used to amplify sample nucleic acid and generate fullhairpin amplicons (e.g., a full hairpin comprising the sequencing primerbinding site (e.g., R1), the copied sample nucleic acid, and thecomplement to the sequencing primer binding site hybridized with thesequencing primer binding site (e.g., R1c)—3404 in FIG. 34B) via theamplification scheme depicted in FIGS. 15A-15G. Upon generation of thefull hairpin amplicons 3404, the full hairpin amplicons can haveadditional sequences (e.g., a sequence comprising a P5 sequence and abarcode sequence) 3405 added, for example, via ligation 3406.

In some cases, primers (e.g., oligonucleotides comprising a randomN-mer) used to generate full hairpin amplicons may be covalentlymodified to comprise an additional sequence via, for example, a linker(e.g., a linker not comprising nucleic acid or a linker comprisingnucleic acid that does not participate in amplification). In some cases,the linker may be polyethylene glycol or a carbon-based linker. Fullhairpin amplicons generated from the primers (e.g., via an amplificationscheme depicted in FIGS. 15A-15G), thus, can also be covalently linkedto the additional sequence via the linker. The attached sequence canthen be ligated to the full hairpin amplicon to generate a partialhairpin species. An example of a full hairpin amplicon 3409 comprisingan additional sequence 3408 via a linker 3407 is shown in FIG. 34C.Following full hairpin generation, the additional sequence 3408 can beligated to the full hairpin amplicon 3409 such that a partial hairpinspecies (3410) comprising the additional sequence 3408 can be generated.

Targeted N-mers and Targeted Amplification

In addition to random amplification schemes, barcode constructs (e.g.,oligonucleotides comprising a barcode sequence and an N-mer for priminga sample nucleic acid) comprising targeted priming sequences (e.g., atargeted N-mer) and targeted amplification schemes are also envisioned.Targeted amplification schemes may be useful, for example, in detectinga particular gene or sequence of interest via sequencing methods, may beuseful in detecting a particular type of nucleic acid, may be useful indetecting the a particular strand of nucleic acid comprising a sequence,and combinations thereof. In general, targeted amplification schemesrely on targeted primers to complete amplification of a particularnucleic acid sequence. In some examples, PCR methods may be used fortargeted amplification, via the use of primers targeted toward aparticular gene sequence of interest or a particular sequence upstreamof a particular gene sequence of interest, such that the particular genesequence of interest is amplified during PCR.

The PHASE amplification reaction described above may also be modifiedsuch that target amplification of sample nucleic acid is achieved.Barcode constructs comprising a targeted priming sequence (e.g., atargeted N-mer), rather than a random sequence (e.g., a random N-mer),as described above, may be used to prime a specific sequence duringPHASE amplification. The specific sequence, for example, may be aparticular gene sequence of interest such that generation of ampliconsis indicative of the sequence's presence. Or, the specific sequence maybe a sequence known to be upstream from a particular gene sequence ofinterest. Such constructs may be generated, and, if desired, coupled tobeads, using any of the methods described herein, including limitingdilution schemes depicted in FIGS. 4A-4N and the combinatorial plateschemes described elsewhere herein.

For example, as described previously with respect to FIGS. 4A-4N, aconstruct comprising a primer 403 (e.g., P5), a barcode sequence 408,and a read primer binding site (e.g., R1) 415 can be generated (seeFIGS. 4A-4H). As shown in FIG. 4I, an additional sequence 413 can beadded (optionally in bulk) to the construct via primer comprising asequence 412 complementary to read primer binding site 415. Sequence 413may serve as a targeted sequence (e.g., a targeted N-mer) such that thetargeted sequence corresponds to a particular target sequence ofinterest. The construct may also comprise an oligonucleotide blocker, asdescribed elsewhere herein, in order to ensure that only the targetedsequence, and not other sequence portions of the construct, primes thesample nucleic acid. Upon entry of the completed construct into a PHASEreaction with sample nucleic acid, for example, the targeted constructmay prime the sample nucleic acid (e.g., at the desired sequence site)and the amplification reaction can be initiated to generate partialhairpins from the sample nucleic acid as described above. In some cases,a combination of targeted N-mer primers and random N-mer primers areused to generate partial hairpin amplicons. In some cases, targetedamplification may be useful in controlling the size (e.g., sequencelength) of partial hairpin amplicons that are generated duringamplification for a particular target.

In some cases, a plurality of constructs comprising a barcode sequenceand a targeted N-mer may be coupled to a bead (e.g., a gel bead). Insome cases, the plurality of constructs may comprise an identicalbarcode sequence and/or an identical targeted N-mer sequence. In somecases, the targeted N-mer sequence may vary amongst individualconstructs of the plurality such that a plurality of target sequences ona sample nucleic acid may be primed via the various targeted N-mers. Asdescribed above, the beads may be partitioned (e.g., in fluidicdroplets) with sample nucleic acid, the bead(s) in each partitiondegraded to release the coupled constructs into the partition, and thesample nucleic acid amplified via the targeted N-mer of the constructs.Post processing (e.g., addition of additional sequences (e.g., P7, R2),addition of a sample index, etc.) of the generated amplicons may beachieved with any method described herein, including bulk amplificationmethods (e.g., bulk PCR) and bulk ligation.

In a partition, constructs comprising a barcode sequence and a targetedN-mer may be coupled to a bead, may be free in solution (e.g., free inthe aqueous interior of a fluidic droplet), or both. Moreover, apartition may comprise both targeted constructs (e.g., constructscomprising a targeted N-mer sequence) and non-targeted constructs (e.g.,constructs comprising a random N-mer sequence). Each of the targeted andnon-targeted constructs may be coupled to a bead, one of the two may becoupled to a bead, and either construct may also be in solution within apartition.

Where each type of construct is present in a partition, both targetedand non-targeted amplification of sample nucleic acids may take place.For example, with respect to a PHASE amplification reaction, a targetedbarcode construct may be used to initially prime and extend a samplenucleic acid. In general, these steps correspond to the first cycle ofPHASE amplification described above with respect to FIGS. 15A-15C,except that the targeted construct is used for initial priming. Theextension products can then be primed with a barcode constructcomprising a random N-mer such that a partial hairpin is generated,these steps corresponding to the second cycle of PHASE described abovewith respect to FIGS. 15D-15F. Amplification can continue for additionalrounds (e.g., FIG. 15G) until the desired number of rounds are complete.Post processing (e.g., addition of additional sequences (e.g., P7, R2),addition of a sample index, etc.) of the generated partial hairpinamplicons may be achieved with any method described herein, includingbulk amplification methods (e.g., bulk PCR) and bulk ligation.

Moreover, targeted barcode constructs may be generated such that theconstruct's targeted N-mer is directed toward nucleic acid species otherthan DNA, such as, for example, an RNA species. In some cases, thetargeted barcode construct's targeted N-mer may be directed toward aparticular RNA sequence, such as, for example, a sequence correspondingto transcribed gene or other sequence on a messenger RNA (mRNA)transcript. In some cases, sequencing of barcoded products generatedfrom RNA (e.g., an mRNA) may aid in determining the expression level ofa gene transcribed by the RNA. In some cases, the targeted N-mer may bea poly-thymine (e.g., poly-T sequence) sequence capable of hybridizingwith a poly-adenine (poly-A sequence) that can, for example, be found atthe 3′ end of an mRNA transcript. Upon priming of an mRNA with atargeted barcode construct comprising a poly-T sequence viahybridization of the barcode construct's poly-T sequence with the mRNA'spoly-A sequence, the targeted barcode construct can be extended via areverse transcription reaction to generate a complementary DNA (cDNA)product comprising the barcode construct. In some cases, a targetedbarcode construct comprising a poly-T targeted N-mer may also comprisean oligonucleotide blocker as described elsewhere herein, such that onlythe poly-T sequence hybridizes with RNA.

Targeted barcode constructs to RNA species may also be useful ingenerating partial hairpin amplicons via, for example, a PHASEamplification reaction. For example, a targeted barcode constructcomprising a poly-T sequence can hybridize with an mRNA via its poly-Asequence. The targeted barcode construct can be extended via a reversetranscription reaction (e.g., via the action of a reverse transcriptase)such that a cDNA comprising the barcode construct is generated. Thesesteps can correspond to the first cycle of PHASE amplification describedabove with respect to FIGS. 15A-15C, except that reverse transcriptionis used to generate the extension product. Following reversetranscription (e.g., a first PHASE cycle), a barcode constructcomprising a random N-mer may prime the extension products such that apartial hairpin is generated as described above with respect to FIGS.15D-15F. Amplification can continue for additional rounds (e.g., FIG.15G) until the desired number of rounds are complete.

In some cases, a plurality of targeted constructs comprising a barcodesequence and a targeted N-mer comprising a poly-T sequence may becoupled to a bead (e.g., a gel bead). In some cases, the plurality ofconstructs may comprise an identical barcode sequence. The beads may bepartitioned (e.g., in fluidic droplets) with sample nucleic acidcomprising RNA, the bead(s) in each partition degraded to release thecoupled constructs into the partition, and the sample RNA captured viathe targeted N-mer of the constructs. Partitions may also comprisebarcode constructs (e.g., with barcode sequences identical to thetargeted constructs) that comprise a random N-mer. In a firstamplification cycle, extension of the targeted constructs can occur viareverse transcription within each partition, to generate extensionproducts comprising the targeted construct. The extension products ineach partition can then be primed with the barcode constructs comprisingthe random N-mer to generate partial hairpin amplicons as describedabove with respect to FIGS. 15A-15G. Post processing (e.g., addition ofadditional sequences (e.g., P7, R2), addition of a sample index, etc.)of the generated amplicons may be achieved with any method describedherein, including bulk amplification methods (e.g., bulk PCR) and bulkligation.

In some cases, reverse transcription of RNA in a sample may also be usedwithout the use of a targeted barcode construct. For example, samplenucleic acid comprising RNA may be first subject to a reversetranscription reaction with other types of reverse transcription primerssuch that cDNA is generated from the RNA. The cDNA that is generated maythen undergo targeted or non-targeted amplification as described herein.For example, sample nucleic acid comprising RNA may be subject to areverse transcription reaction such that cDNA is generated from the RNA.The cDNA may then enter a PHASE amplification reaction, using a barcodeconstruct with a random N-mer as described above with respect to FIGS.15A-15G, to generate partial hairpin amplicons comprising theconstruct's barcode sequence. Post processing (e.g., addition ofadditional sequences (e.g., P7, R2), addition of a sample index, etc.)of the generated partial hairpin amplicons may be achieved with anymethod described herein, including bulk amplification methods (e.g.,bulk PCR) and bulk ligation.

Targeted barcode constructs may also be generated toward specificsequences (e.g., gene sequences) on specific strands of a nucleic acidsuch that strandedness information is retained for sequencer-readyproducts generated for each strand. For example, a sample nucleic maycomprise double stranded nucleic acid (e.g., double-stranded DNA), suchthat each strand of nucleic acid comprises one or more different targetgene sequences. Complementary DNA strands can comprise different genesequences due to the opposite 5′ to 3′ directionalities and/or basecomposition of each strand. Targeted barcode constructs can be generatedfor each strand (based on 5′ to 3′ directionality of the strand) basedon the targeted N-mer and configuration of the barcode construct.Example sets of targeted barcode constructs directed to forward andreverse strands of a double-stranded sample nucleic acid are shown inFIG. 28A.

Example sets 2801 and 2802 of targeted barcode constructs each targetedto either of a forward (2801) strand and reverse (2802) strand of adouble-stranded sample nucleic acid are shown in FIG. 28A. Set 2801comprises targeted barcode constructs 2803 and 2804 comprising a P5sequence, a barcode sequence, and a targeted N-mer to either of a firsttarget sequence (2803) or a second target sequence (2804). Set 2802comprises targeted barcode constructs 2805 and 2806 comprising a P5sequence, a barcode sequence, and a targeted N-mer to either of thefirst target sequence (2805) and the second target sequence (2806). Eachconstruct can also comprise any additional sequences between the barcodeand the targeted N-mer (indicated by an arrow in each construct shown inFIG. 28A).

The barcode constructs in set 2801 are configured to prime theirrespective target sequences on the forward strand of the double-strandedsample nucleic acid. The barcode constructs of set 2802 are configuredto prime their respective target sequences on the reverse strand of thedouble-stranded sample nucleic acid. As shown, the targeted barcodeconstructs in each set are configured in opposite directionalitycorresponding to the opposite directionality of forward and reversestrands of the double-stranded sample nucleic acid. Each barcodeconstruct can prime its respective target sequence on its respectivestrand of sample nucleic acid to generate barcoded amplicons via anamplification reaction, such as any amplification reaction describedherein.

Additional sequences can be added to barcoded amplicons usingamplification methods described herein, including bulk amplification,bulk ligation, or a combination thereof. Example sets of primers thatmay be used to add a sample index and P7 sequence to amplicons generatedfrom the targeted barcode constructs in FIG. 28A are shown in FIG. 28B.Primer set 2808 corresponds to targeted barcode construct set 2801(e.g., targeted barcode construct 2803 corresponds to primer 2811,targeted barcode construct 2804 corresponds to primer 2812) and primerset 2808 corresponds to targeted barcode construct set 2801 (e.g.,targeted barcode construct 2505 corresponds to primer 2809, targetedbarcode construct 2806 corresponds to primer 2810). Each primer canprime its respective target sequence on its respective strand and bulkamplification (e.g., bulk PCR) initiated to generate sequencer-readyconstructs that include the P7 and sample index sequences in analogousfashion to bulk amplification methods described elsewhere herein. Basedon the configuration and directionality of the various components ofeach sequencer-ready construct (e.g., P5, barcode, targeted N-mer,sample insert, etc.), the strand from which the sequencer-ready productis generated can be determined/is retained.

Libraries of barcode constructs (e.g., targeted barcode constructs) maybe generated for both forward and reverse strands of a double strandednucleic acid. For example, two libraries of beads (e.g., gel beads)comprising targeted barcode constructs may be generated using methodsdescribed herein, such that one library comprises targeted barcodeconstructs for forward strands of sample nucleic acids and the otherlibrary comprises targeted barcode constructs for reverse strands ofsample nucleic acids. In some cases, each library may comprise beadseach comprising an identical targeted N-mer. In some cases, each librarymay comprise two or more sets of beads, with each bead in a setcomprising an identical targeted N-mer (e.g., a targeted N-mer targetedtoward a particular gene) and different sets comprising differenttargeted N-mers. In some cases, the two libraries may be combined suchthat a library of forward strand and reverse strand beads is generated.

For example, a library can comprise two types of forward strand beadsand two types of reverse strand beads, for a total of four types ofbeads. Each bead in the library may comprise a unique barcode sequence.One type of the forward strand beads and one type of the reverse strandbeads may comprise targeted N-mers corresponding to a target sequence(e.g., a target gene sequence). For example, one type of forward strandbeads may comprise a targeted barcode construct as shown in 2803 in FIG.28A and one type of reverse strand beads may comprise a targeted barcodeconstruct as shown in 2805 in FIG. 28A. Analogously, the second type offorward strand beads may comprise a targeted barcode construct as shownin 2804 in FIG. 28A and one type of reverse strand beads may comprise atargeted barcode construct as shown in 2806 in FIG. 28A.

A barcode library comprising forward strand and reverse strand beads(e.g., gel beads), with each bead comprising a unique barcode sequencemay be partitioned to barcode sample nucleic acids as describedelsewhere herein. For example, the mixed library of two types of forwardstrand and two types of reverse strand beads described above may bepartitioned with a sample nucleic acid (e.g., genomic DNA) and any otherdesired reagents (e.g., reagents necessary for amplification of thesample nucleic acid, a reducing agent). The partitions may be, forexample, fluidic droplets such as droplets of an emulsion. In general,each partition may comprise a bead (e.g., a forward strand bead or areverse strand bead) coupled to a targeted barcode construct comprisinga unique barcode sequence and a targeted N-mer. In some cases, though,one or more of the partitions may comprise multiple beads of the sametype or of different types. The targeted barcode constructs may bereleased from the bead (e.g., via degradation of the bead—for example,via a reducing agent in cases where the bead is a gel bead comprisingdisulfide bonds) in the partition and allowed to prime their targetsequence on their respective strand (e.g., forward strand or reversestrand) of sample nucleic acid.

A first product strand synthesis may take place in each partition viaextension of the hybridized targeted barcode construct, via, forexample, linear amplification of the sample nucleic acid. Additionalrounds of linear amplification of the sample nucleic acid with thetargeted barcode construct, for example, may be used to generateadditional copies of the first product strand. First product strands maythen be removed from the partitions (e.g., in cases where the partitionsare droplets of an emulsion, the emulsion may be broken to release firstproducts) and pooled. The first products may be washed to removetargeted barcode constructs and any other waste products. In some cases,an optional double-stranded digestion may be completed to digest samplenucleic acid and remove it from the first product strands.

Next, the first product strands may be subject to bulk amplification toadd additional sequences (e.g., P7, a sample index, etc.) to the firstproduct strands, resulting in the generation of second product strands.The bulk amplification reaction mixture may comprise a plurality ofprimers, with each primer in the plurality corresponding to one of thebead types (and, thus, type of targeted barcode construct) used togenerate the first products strands. For the example library comprisingtwo types of forward strand beads and two types of reverse strand beadsdescribed above, primers shown as 2809, 2810, 2811, and 2812 in FIG. 28Bmay be used to add additional sample index and P7 sequences to firstproduct strands generated from targeted barcode constructs 2803, 2804,2805, and 2806 respectively via bulk amplification. Second productstrands may then be washed to remove primers from the reaction mixture.Fresh primers (e.g., primers comprising P5 and P7 for the exampledescribed above) may then be added one or more additional rounds ofamplification (e.g., via PCR) to generate final, sequencer-readyproducts. Thus, final products can comprise the original targetedbarcode construct, the strand of sample nucleic acid amplified, and theadditional sequences (e.g., P7, sample index) added to first productstrands.

Methods described herein may be useful in whole genome amplification. Insome embodiments of whole genome amplification, a random primer (e.g., arandom N-mer sequence) can be hybridized to a genomic nucleic acid. Therandom primer can be a component of a larger oligonucleotide that mayalso include a universal nucleic acid sequence (including any type ofuniversal nucleic acid sequence described herein) and a nucleic acidbarcode sequence. In some cases, the universal nucleic acid sequence maycomprise one or more uracil containing nucleotides. Moreover, in somecases, the universal nucleic acid sequence may comprise a segment of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more nucleotides that do not comprise uracil. The random primercan be extended (e.g., in a primer extension reaction or any othersuitable type of nucleic acid amplification reaction) to form anamplified product.

As described elsewhere herein, the amplified product may undergo anintramolecular hybridization reaction to form a hairpin molecule suchas, for example, a partial hairpin molecule. In some cases, whole genomeamplification may occur in the presence of an oligonucleotide blocker(also referred to as a blocker oligonucleotide elsewhere herein) thatmay or may not comprise a blocker moiety (e.g., C3 spacer (/3SpC3/),Dideoxy-C(/3ddC/), 3′ phosphate, or any other type of blocker moietydescribed elsewhere herein). Furthermore, the oligonucleotide blockermay be capable of hybridizing to at least a portion of the universalnucleic acid sequence or any other part of an oligonucleotide comprisingthe random primer.

In some embodiments of whole genome amplification, a genomic component(e.g., a chromosome, genomic nucleic acid such as genomic DNA, a wholegenome of an organism, or any other type of genomic component describedherein) may be fragmented in a plurality of first fragments. The firstfragments can be co-partitioned into a plurality of partitions with aplurality of oligonucleotides. The oligonucleotides in each of thepartitions may comprise a primer sequence (including a type of primersequence described elsewhere herein) and a common sequence (e.g., abarcode sequence). Primer sequences in each partition can then beannealed to a plurality of different regions of the first fragmentswithin each partition. The primer sequences can then be extended alongthe first fragments to produce amplified first fragments within eachpartition of the plurality of partitions. The amplified first fragmentswithin the partitions may comprise any suitable coverage (as describedelsewhere herein) of the genomic component. In some cases, the amplifiedfirst fragments within the partitions may comprise at least 1× coverage,at least 2×coverage, at least 5× coverage, at least 10× coverage, atleast 20× coverage, at least 40× coverage, or greater coverage of thegenomic component.

VII. Digital Processor

The methods, compositions, devices, and kits of this disclosure may beused with any suitable processor, digital processor or computer. Thedigital processor may be programmed, for example, to operate anycomponent of a device and/or execute methods described herein. Thedigital processor may be capable of transmitting or receiving electronicsignals through a computer network, such as for example, the Internetand/or communicating with a remote computer. One or more peripheraldevices such as screen display, printer, memory, data storage, and/orelectronic display adaptors may be in communication with the digitalprocessor. One or more input devices such as keyboard, mouse, orjoystick may be in communication with the digital processor. The digitalprocessor may also communicate with detector such that the detectorperforms measurements at desired or otherwise predetermined time pointsor at time points determined from feedback received from pre-processingunit or other devices.

A conceptual schematic for an example control assembly is shown in FIG.18. A computer, serves as the central hub for control assembly. Thecomputer is in communication with a display, one or more input devices(e.g., a mouse, keyboard, camera, etc.), and optionally a printer. Thecontrol assembly, via its computer, is in communication with one or moredevices: optionally a sample pre-processing unit, one or more sampleprocessing units (such as a sequence, thermocycler, or microfluidicdevice), and optionally a detector. The control assembly may benetworked, for example, via an Ethernet connection. A user may provideinputs (e.g., the parameters necessary for a desired set of nucleic acidamplification reactions or flow rates for a microfluidic device) intothe computer, using an input device. The inputs are interpreted by thecomputer, to generate instructions. The computer communicates suchinstructions to the optional sample pre-processing unit, the one or moresample processing units, and/or the optional detector for execution.

Moreover, during operation of the optional sample pre-processing unit,one or more sample processing units, and/or the optional detector, eachdevice may communicate signals back to computer. Such signals may beinterpreted and used by computer to determine if any of the devicesrequire further instruction. The computer may also modulate the samplepre-processing unit such that the components of a sample are mixedappropriately and fed, at a desired or otherwise predetermined rate,into the sample processing unit (such as the microfluidic device).

The computer may also communicate with a detector such that the detectorperforms measurements at desired or otherwise predetermined time pointsor at time points determined from feedback received from pre-processingunit or sample processing unit. The detector may also communicate rawdata obtained during measurements back to the computer for furtheranalysis and interpretation.

Analysis may be summarized in formats useful to an end user via adisplay and/or printouts generated by a printer. Instructions orprograms used to control the sample pre-processing unit, the sampleprocessing unit, and/or the detector; data acquired by executing any ofthe methods described herein; or data analyzed and/or interpreted may betransmitted to or received from one or more remote computers, via anetwork, which, for example, could be the Internet.

In some embodiments, the method of bead formation may be executed withthe aid of a digital processor in communication with a dropletgenerator. The digital processor may control the speed at which dropletsare formed or control the total number of droplets that are generated.In some embodiments, the method of attaching samples to barcoded beadsmay be executed with the aid of a digital processor in communicationwith the microfluidic device. Specifically, the digital processor maycontrol the volumetric amount of sample and/or beads injected into theinput channels and may also control the flow rates within the channels.In some embodiments, the method of attaching oligonucleotides, primers,and the like may be executed with the aid of a digital processor incommunication with a thermocycler or other programmable heating element.Specifically, the digital processor may control the time and temperatureof cycles during ligation or amplification. In some embodiments, themethod of sequencing a sample may be executed with the aid of a digitalprocessor in communication with a sequencing device.

VIII. Kits

In some cases, this disclosure provides a kit comprising a microfluidicdevice, a plurality of barcoded beads, and instructions for utilizingthe microfluidic device and combining barcoded beads with customersample to create fluidic droplets containing both. As specifiedthroughout this disclosure, any suitable sample may be incorporated intothe fluidic droplets. As described throughout this disclosure, a beadmay be designed to be degradable or non-degradable. In this case, thekit may or may not include a reducing agent for bead degradation.

In some cases, this disclosure provides a kit comprising a plurality ofbarcoded beads, suitable amplification reagents, e.g., optionallyincluding one or more of polymerase enzymes, nucleoside triphosphates ortheir analogues, primer sequences, buffers, and the like, andinstructions for combining barcoded beads with customer sample. Asspecified throughout this disclosure, any suitable sample may be used.As specified throughout this disclosure, the amplification reagents mayinclude a polymerase that will not accept or process uracil-containingtemplates. A kit of this disclosure may also provide agents to form anemulsion, including an oil and surfactant.

IX. Applications Barcoding Sample Materials

The methods, compositions and systems described herein are particularlyuseful for attaching barcodes, and particularly barcode nucleic acidsequences, to sample materials and components of those sample materials.In general, this is accomplished by partitioning sample materialcomponents into separate partitions or reaction volumes in which areco-partitioned a plurality of barcodes, which are then attached tosample components within the same partition.

In an exemplary process, a first partition is provided that includes aplurality of oligonucleotides (e.g., nucleic acid barcode molecules)that each comprise a common nucleic acid barcode sequence. The firstpartition may comprise any of a variety of portable partitions, e.g., abead (e.g., a degradable bead, a gel bead), a droplet (e.g., an aqueousdroplet in an emulsion), a microcapsule, or the like, to which theoligonucleotides are releasably attached, releasably coupled, or arereleasably associated. Moreover, any suitable number of oligonucleotidesmay be included in the first partition, including numbers ofoligonucleotides per partition described elsewhere herein. For example,the oligonucleotides may be releasably attached to, releasably coupledto, or releasably associated with the first partition via a cleavablelinkage such as, for example, a chemically cleavable linkage (e.g., adisulfide linkage, or any other type of chemically cleavable linkagedescribed herein), a photocleavable linkage, and/or a thermallycleavable linkage. In some cases, the first partition may be a bead andthe bead may be a degradable bead (e.g., a photodegradable bead, achemically degradable bead, a thermally degradable bead, or any othertype of degradable bead described elsewhere herein). Moreover, the beadmay comprise chemically-cleavable cross-linking (e.g., disulfidecross-linking) as described elsewhere herein.

The first partition is then co-partitioned into a second partition, witha sample material, sample material component, fragment of a samplematerial, or a fragment of a sample material component. The samplematerial (or component or fragment thereof) may be any appropriatesample type, including the example sample types described elsewhereherein. In cases where a sample material or component of a samplematerial comprises one or more nucleic acid fragments, the one or morenucleic acid fragments may be of any suitable length, including, forexample, nucleic acid fragment lengths described elsewhere herein. Thesecond partition may include any of a variety of partitions, includingfor example, wells, microwells, nanowells, tubes or containers, or inpreferred cases droplets (e.g., aqueous droplets in an emulsion) ormicrocapsules in which the first partition may be co-partitioned. Insome cases, the first partition may be provided in a first aqueous fluidand the sample material, sample material component, or fragment of asample material component may be provided in a second aqueous fluid.During co-partitioning, the first aqueous fluid and second aqueous fluidmay be combined within a droplet within an immiscible fluid. In somecases, the second partition may comprise no more than one firstpartition. In other cases, the second partition may comprise no morethan one, two, three, four, five, six, seven, eight, nine, or ten firstpartitions. In other cases, the second partition may comprise at leastone, two, three, four, five, six, seven, eight, nine, ten, or more firstpartitions.

Once co-partitioned, the oligonucleotides comprising the barcodesequences may be released from the first partition (e.g., viadegradation of the first partition, cleaving a chemical linkgage betweenthe oligonucleotides and the first partition, or any other suitable typeof release, including types of release described elsewhere herein) intothe second partition, and attached to the sample componentsco-partitioned therewith. In some cases, the first partition maycomprise a bead and the crosslinking of the bead may comprise adisulfide linkage. In addition, or as an alternative, theoligonucleotides may be linked to the bead via a disulfide linkage. Ineither case, the oligonucleotides may be released from the firstpartition by exposing the first partition to a reducing agent (e.g.,DTT, TCEP, or any other exemplary reducing agent described elsewhereherein).

As noted elsewhere herein, attachment of the barcodes to samplecomponents includes the direct attachment of the barcodeoligonucleotides to sample materials, e.g. through ligation,hybridization, or other associations. Additionally, in many cases, forexample, in barcoding of nucleic acid sample materials (e.g., templatenucleic acid sequences, template nucleic acid molecules), components orfragments thereof, such attachment may additionally comprise use of thebarcode containing oligonucleotides that also comprise as primingsequences. The priming sequence can be complementary to at least aportion of a nucleic acid sample material and can be extended along thenucleic acid sample materials to create complements to such samplematerials, as well as at least partial amplification products of thosesequences or their complements.

In another exemplary process, a plurality of first partitions can beprovided that comprise a plurality of different nucleic acid barcodesequences. Each of the first partitions can comprise a plurality ofnucleic acid barcode molecules having the same nucleic acid barcodesequence associated therewith. Any suitable number of nucleic acidbarcode molecules may be associated with each of the first partitions,including numbers of nucleic acid barcode molecules per partitiondescribed elsewhere herein. The first partitions may comprise anysuitable number of different nucleic acid barcode sequences, including,for example, at least about 2, 10, 100, 500, 1000, 5000, 10000, 50000,100000, 500000, 1000000, 5000000, 10000000, 50000000, or 1000000000, ormore different nucleic acid barcode sequences.

In some cases, the plurality of first partitions may comprise aplurality of different first partitions where each of the differentfirst partitions comprises a plurality of releasably attached,releasably coupled, or releasably associated oligonucleotides comprisinga common barcode sequence, with the oligonucleotides associated witheach different first partitions comprising a different barcode sequence.The number of different first partitions may be, for example, at leastabout 2, 10, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000,1000000, 5000000, 10000000, 50000000, or 1000000000, or more differentfirst partitions.

The first partitions may be co-partitioned with sample materials,fragments of a sample material, components of a sample material, orfragments of a component(s) of a sample material into a plurality ofsecond partitions. In some cases, a subset of the second partitions maycomprise the same nucleic acid barcode sequence. For example, at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more ofthe second partitions may comprise the same nucleic acid barcodesequence. Moreover, the distribution of first partitions per secondpartition may also vary according to, for example, occupancy ratesdescribed elsewhere herein. In cases where the plurality of firstpartitions comprises a plurality of different first partitions, eachdifferent first partition may be disposed within a separate secondpartition.

Following co-partitioning, the nucleic acid barcode molecules associatedwith the first partitions can be released into the plurality of secondpartitions. The released nucleic acid barcode molecules can then beattached to the sample materials, sample material components, fragmentsof a sample material, or fragments of sample material components, withinthe second partitions. In the case of barcoded nucleic acid species(e.g., barcoded sample nucleic acid, barcoded template nucleic acid,barcoded fragments of one or more template nucleic acid sequences,etc.), the barcoded nucleic acid species may be sequenced as describedelsewhere herein.

In another exemplary process, an activatable nucleic acid barcodesequence may be provided and partitioned with one or more samplematerials, components of a sample material, fragments of a samplematerial, or fragments of a component(s) of a sample material into afirst partition. With the first partition, the activatable nucleic acidbarcode sequence may be activated to produce an active nucleic acidbarcode sequence. The active nucleic acid barcode sequence can then beattached to the one or more sample materials, components of a samplematerial, fragments of a sample material, or fragments of a component(s)of a sample material.

In some cases, the activatable nucleic acid barcode sequence may becoupled to a second partition that is also partitioned in the firstpartition with the activatable nucleic acid barcode sequence. Asdescribed elsewhere herein, an activatable nucleic acid barcode sequencemay be activated by releasing the activatable nucleic acid barcodesequence from an associated partition (e.g., a bead). Thus, in caseswhere an activatable nucleic acid barcode sequence is associated with asecond partition (e.g., a bead) that is partitioned in a first partition(e.g., a fluidic droplet), the activatable nucleic acid barcode sequencemay be activated by releasing the activatable nucleic acid barcodesequence from its associated second partition. In addition, or as analternative, an activatable barcode may also be activated by removing aremovable blocking or protecting group from the activatable nucleic acidbarcode sequence.

In another exemplary process, a sample of nucleic acids may be combinedwith a library of barcoded beads (including types of beads describedelsewhere herein) to form a mixture. In some cases, the barcodes of thebeads may, in addition to a barcode sequence, each comprise one or moreadditional sequences such as, for example, a universal sequence and/or afunctional sequence (e.g., a random N-mer or a targeted N-mer, asdescribed elsewhere herein). The mixture may be partitioned into aplurality of partitions, with at least a subset of the partitionscomprising at most one barcoded bead. Within the partitions, thebarcodes may be released from the beads, using any suitable route,including types of release described herein. A library of barcoded beadsmay be generated via any suitable route, including the use of methodsand compositions described elsewhere herein. In some cases, the sampleof nucleic acids may be combined with the library of barcoded beadsand/or the resulting mixture partitioned with the aid of a microfluidicdevice, as described elsewhere herein. In cases where the releasedbarcodes also comprise a primer sequence (e.g., such as a targeted N-meror a random N-mer as described elsewhere herein), the primer sequencesof the barcodes may be hybridize with the sample nucleic acids and, ifdesired, an amplification reaction can be completed in the partitions.

Polynucleotide Sequencing

Generally, the methods and compositions provided herein are useful forpreparation of oligonucleotide fragments for downstream applicationssuch as sequencing. In particular, these methods, compositions andsystems are useful in the preparation of sequencing libraries.Sequencing may be performed by any available technique. For example,sequencing may be performed by the classic Sanger sequencing method.Sequencing methods may also include: high-throughput sequencing,pyrosequencing, sequencing-by-ligation, sequencing by synthesis,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), massively-parallel sequencing, clonal singlemolecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing,primer walking, and any other sequencing methods known in the art.

For example, a plurality of target nucleic acid sequences may besequenced by providing a plurality of target nucleic sequences andseparating the target nucleic acid sequences into a plurality ofseparate partitions. Each of the separate partitions can comprise one ormore target nucleic acid sequences and a plurality of oligonucleotides.The separate partitions may comprise any suitable number of differentbarcode sequences (e.g., at least 1,000 different barcode sequences, atleast 10,000 different barcode sequences, at least 100,000 differentbarcode sequences, at least 1,000,000 different barcode sequences, atleast 10,000,000 different barcode sequences, or any other number ofdifferent barcode sequences as described elsewhere herein). Moreover,the oligonucleotides in a given partition can comprise a common barcodesequence. The oligonucleotides and associated common barcode sequence ina given partition can be attached to fragments of the one or more targetnucleic acids or to copies of portions of the target nucleic acidsequences within the given partition. Following attachment, the separatepartitions can then be pooled. The fragments of the target nucleic acidsor the copies of the portions of the target nucleic acids and attachedbarcode sequences can then be sequenced.

In another example, a plurality of target nucleic acid sequences may besequenced by providing the target nucleic acid sequences and separatingthem into a plurality of separate partitions. Each partition of theplurality of separate partitions can include one or more of the targetnucleic acid sequences and a bead having a plurality of attachedoligonucleotides. The oligonucleotides attached to a given bead maycomprise a common barcode sequence. The oligonucleotides associated witha bead can be attached to fragments of the target nucleic acid sequencesor to copies of portions of the target nucleic acid sequences within agiven partition, such that the fragments or copies of the givenpartition are also attached to the common barcode sequence associatedwith the bead. Following attachment of the oligonucleotides to thefragments of the target nucleic acid sequences or the copies of theportions of the target nucleic acid sequences, the separate partitionscan then be pooled. The fragments of the target nucleic acid sequencesor the copies of the portions of the target nucleic acid sequences andany attached barcode sequences can then be sequenced (e.g., using anysuitable sequencing method, including those described elsewhere herein)to provide barcoded fragment sequences or barcoded copy sequences. Thebarcoded fragment sequences or barcoded copy sequences can be assembledinto one or more contiguous nucleic acid sequence based, in part, upon abarcode portion of the barcoded fragment sequences or barcoded copysequences.

In some cases, varying numbers of barcoded-oligonucleotides aresequenced. For example, in some cases about 30%-90% of thebarcoded-oligonucleotides are sequenced. In some cases, about 35%-85%,40%-80%, 45%-75%, 55%-65%, or 50%-60% of the barcoded-oligonucleotides sare sequenced. In some cases, at least about 30%, 40%, 50%, 60%, 70%,80%, or 90% of barcoded-oligonucleotides are sequenced. In some cases,less than about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of thebarcoded-oligonucleotides are sequenced.

In some cases, sequences from fragments are assembled to providesequence information for a contiguous region of the original targetpolynucleotide that may be longer than the individual sequence reads.Individual sequence reads may be about 10-50, 50-100, 100-200, 200-300,300-400, or more nucleotides in length. Examples of sequence assemblymethods include those set forth in U.S. Provisional Patent ApplicationNo. 62/017,589 (Attorney Docket No. 43487-729.101), filed of even dateherewith.

The identities of the barcodes may serve to order the sequence readsfrom individual fragments as well as to differentiate betweenhaplotypes. For example, when combining individual sample fragments andbarcoded beads within fluidic droplets, parental polynucleotidefragments may be separated into different droplets. With an increase inthe number of fluidic droplets and beads within a droplet, thelikelihood of a fragment from both a maternal and paternal haplotypecontained within the same fluidic droplet associated with the same beadmay become negligibly small. Thus, sequence reads from fragments in thesame fluidic droplet and associated with the same bead may be assembledand ordered.

In at least one example, the present disclosure provides nucleic acidsequencing methods, systems compositions, and combinations of these thatare useful in providing myriad benefits in both sequence assembly andread-length equivalent, but do so with very high throughput and reducedsample preparation time and cost.

In general, the sequencing methods described herein provide for thelocalized tagging or barcoding of fragments of genetic sequences. Bytagging fragments that derive from the same location within a largergenetic sequence, one can utilize the presence of the tag or barcode toinform the assembly process as alluded to above. In addition, themethods described herein can be used to generate and barcode shorterfragments from a single, long nucleic acid molecule. Sequencing andassembly of these shorter fragments provides a long read equivalentsequence, but without the need for low throughput longer read-lengthsequencing technologies.

FIG. 39 provides a schematic illustration of an example sequencingmethod. As shown, a first genetic component 3902 that may comprise, forexample, a chromosome or other large nucleic acid molecule, isfragmented into a set of large first nucleic acid fragments, e.g.,including fragments 3904 and 3906. The fragments of the large geneticcomponent may be non-overlapping or overlapping, and in some cases, mayinclude multifold overlapping fragments, in order to provide for highconfidence assembly of the sequence of the larger component. In somecases, the fragments of the larger genetic component provide 1×, 2×, 5×,10×, 20×, 40× or greater coverage of the larger component.

One or more of the first fragments 3904 is then processed to separatelyprovide overlapping set of second fragments of the first fragment(s),e.g., second fragment sets 3908 and 3910. This processing also providesthe second fragments with a barcode sequence that is the same for eachof the second fragments derived from a particular first fragment. Asshown, the barcode sequence for second fragment set 3908 is denoted by“1” while the barcode sequence for fragment set 3910 is denoted by “2”.A diverse library of barcodes may be used to differentially barcodelarge numbers of different fragment sets. However, it is not necessaryfor every second fragment set from a different first fragment to bebarcoded with different barcode sequences. In fact, in many cases,multiple different first fragments may be processed concurrently toinclude the same barcode sequence. Diverse barcode libraries aredescribed in detail elsewhere herein.

The barcoded fragments, e.g., from fragment sets 3908 and 3910, may thenbe pooled for sequencing. Once sequenced, the sequence reads 3912 can beattributed to their respective fragment set, e.g., as shown inaggregated reads 3914 and 3916, at least in part based upon the includedbarcodes, and optionally, and preferably, in part based upon thesequence of the fragment itself. The attributed sequence reads for eachfragment set are then assembled to provide the assembled sequence forthe first fragments, e.g., fragment sequences 3918 and 3920, which inturn, may be assembled into the sequence 3922 of the larger geneticcomponent.

In accordance with the foregoing, a large genetic component, such as along nucleic acid fragment, e.g., 1, 10, 20, 40, 50, 75, 100, 1000 ormore kb in length, a chromosomal fragment or whole chromosome, or partof or an entire genome (e.g., genomic DNA) is fragmented into smallerfirst fragments. Typically, these fragments may be anywhere from about1000 to about 100000 bases in length. In certain preferred aspects, thefragments will be between about 1 kb and about 100 kb, or between about5 kb and about 50 kb, or from about 10 kb to about 30 kb, and in somecases, between about 15 kb and about 25 kb. Fragmentation of theselarger genetic components may be carried out by any of a variety ofconvenient available processes, including commercially available shearbased fragmenting systems, e.g., Covaris fragmentation systems, sizetargeted fragmentation systems, e.g., Blue Pippin (Sage Sciences),enzymatic fragmentation processes, e.g., using restrictionendonucleases, or the like. As noted above, the first fragments of thelarger genetic component may comprise overlapping or non-overlappingfirst fragments. Although described here as being fragmented prior topartitioning, it will be appreciated that fragmentation may optionallyand/or additionally be performed later in the process, e.g., followingone or more amplification steps, to yield fragments of a desired sizefor sequencing applications.

In preferred aspects, the first fragments are generated from multiplecopies of the larger genetic component or portions thereof, so thatoverlapping first fragments are produced. In preferred aspects, theoverlapping fragments will constitute greater than 1× coverage, greaterthan 2× coverage, greater than 5× coverage, greater than 10× coverage,greater than 20× coverage, greater than 40× coverage, or even greatercoverage of the underlying larger genetic component or portion thereof.The first fragments are then segregated to different reaction volumes.In some cases, the first fragments may be separated so that reactionvolumes contain one or fewer first fragments. This is typicallyaccomplished by providing the fragments in a limiting dilution insolution, such that allocation of the solution to different reactionvolumes results in a very low probability of more than one fragmentbeing deposited into a given reaction volume. However, in most cases, agiven reaction volume may include multiple different first fragments,and can even have 2, 5, 10, 100, 100 or even up to 10,000 or moredifferent first fragments in a given reaction volume. Again, achieving adesired range of fragment numbers within individual reaction volumes istypically accomplished through the appropriate dilution of the solutionfrom which the first fragments originate, based upon an understanding ofthe concentration of nucleic acids in that starting material.

The reaction volumes may include any of variety of different types ofvessels or partitions. For example, the reaction volumes may includeconventional reaction vessels, such as test tubes, reaction wells,microwells, nanowells, or they may include less conventional reactionvolumes, such as droplets within a stabilized emulsion, e.g., a water inoil emulsion system. In preferred aspects, droplets are preferred as thereaction volumes for their extremely high multiplex capability, e.g.,allowing the use of hundreds of thousands, millions, tens of millions oreven more discrete droplet/reaction volumes within a single container.Within each reaction volume, the fragments that are contained thereinare then subjected to processing that both derives sets of overlappingsecond fragments of each of the first fragments, and also provides thesesecond fragments with attached barcode sequences. As will beappreciated, in preferred aspects, the first fragments are partitionedinto droplets that also contain one or more microcapsules or beads thatinclude the members of the barcode library used to generate and barcodethe second fragments.

In preferred aspects, the generation of these second fragments iscarried out through the introduction of primer sequences that includethe barcode sequences and that are capable of hybridizing to portions ofthe first fragment and be extended along the first fragment to provide asecond fragment including the barcode sequence. These primers maycomprise targeted primer sequences, e.g., to derive fragments thatoverlap specific portions of the first fragment, or they may compriseuniversal priming sequences, e.g., random primers, that will primemultiple different regions of the first fragments to create large anddiverse sets of second fragments that span the first fragment andprovide multifold overlapping coverage. These extended primer sequencesmay be used as the second fragments, or they may be further replicatedor amplified. For example, iterative priming against the extendedsequences, e.g., using the same primer containing barcodedoligonucleotides. In certain preferred aspects, the generation of thesecond sets of fragments generates the partial hairpin replicates ofportions of the first fragment, as described elsewhere herein that eachinclude barcode sequences, e.g., for PHASE amplification as describedherein. As noted elsewhere herein, the formation of the partial hairpinis generally desired to prevent repriming of the replicated strand,e.g., making a copy of a copy. As such, the partial hairpin is typicallypreferentially formed from the amplification product during annealing ascompared to a primer annealing to the amplification product, e.g., thehairpin will have a higher Tm than the primer product pair.

The second fragments are generally selected to be of a length that issuitable for subsequent sequencing. For short read sequencingtechnologies, such fragments will typically be from about 50 bases toabout 1000 bases in sequenceable length, from about 50 bases to about900 bases in sequenceable length, from about 50 bases to about 800 basesin sequenceable length, from about 50 bases to about 700 bases insequenceable length, from about 50 bases to about 600 bases insequenceable length, from about 50 bases to about 500 bases insequenceable length, from about 50 bases to about 400 bases insequenceable length, from about 50 bases to about 300 bases insequenceable length, from about 50 bases to about 250 bases insequenceable length, from about 50 bases to about 200 bases insequenceable length, or from about 50 bases to about 100 bases insequenceable length, including the barcode sequence segments, andfunctional sequences that are subjected to the sequencing process.

Once the overlapping, barcoded second fragment sets are generated, theymay be pooled for subsequent processing and ultimately, sequencing. Forexample, in some cases, the barcoded fragments may be subsequentlysubjected to additional amplification, e.g., PCR amplification, asdescribed elsewhere herein. Likewise, these fragments may additionally,or concurrently, be provided with sample index sequences to identify thesample from which collections of barcoded fragments have derived, aswell as providing additional functional sequences for use in sequencingprocesses.

In addition, clean up steps may also optionally be performed, e.g., topurify nucleic acid components from other impurities, to size selectfragment sets for sequencing, or the like. Such clean up steps mayinclude purification and/or size selection upon SPRI beads (such asAmpure® beads, available from Beckman Coulter, Inc.). In some cases,multiple process steps may be carried out in an integrated process whilethe fragments are associated with SPRI beads, e.g., as described inFisher et al., Genome Biol. 2011:12(1):R1 (E-pub Jan. 4, 2011), which isincorporated herein by reference in its entirety for all purposes.

As noted previously, in many cases, short read sequencing technologiesare used to provide the sequence information for the second fragmentsets. Accordingly, in preferred aspects, second fragment sets willtypically comprise fragments that, when including the barcode sequences,will be within the read length of the sequencing system used. Forexample, for Illumina HiSeq® sequencing, such fragments may be betweengenerally range from about 100 bases to about 200 bases in length, whencarrying out paired end sequencing. In some cases, longer secondfragments may be sequenced when accessing only the terminal portions ofthe fragments by the sequencing process.

As noted above with reference to FIG. 39, the sequence reads for thevarious second fragments are then attributed to their respectivestarting nucleic acid segment based in part upon the presence of aparticular barcode sequence, and in some cases, based in part on theactual sequence of the fragment, i.e., a non-barcode portion of thefragment sequence. As will be appreciated, despite being based uponshort sequence data, one can infer that two sequences sharing the samebarcode likely originated from the same longer first fragment sequence,especially where such sequences are otherwise assemble-able into acontiguous sequence segment, e.g., using other overlapping sequencesbearing the common barcode. Once the first fragments are assembled, theymay be assembled into larger sequence segments, e.g., the full lengthgenetic component.

In one exemplary process, one or more fragments of one or more templatenucleic acid sequences may be barcoded using a method described herein.A fragment of the one or more fragments may be characterized based atleast in part upon a nucleic acid barcode sequence attached thereto.Characterization of the fragment may also include mapping the fragmentto its respective template nucleic acid sequence or a genome from whichthe template nucleic acid sequence was derived. Moreover,characterization may also include identifying an individual nucleic acidbarcode sequence and a sequence of a fragment of a template nucleic acidsequence attached thereto.

In some cases, sequencing methods described herein may be useful incharacterizing a nucleic acid segment or target nucleic acid. In someexample methods, a nucleic acid segment may be characterized byco-partitioning the nucleic acid segment and a bead (e.g., including anysuitable type of bead described herein) comprising a plurality ofoligonucleotides that include a common nucleic acid barcode sequence,into a partition (including any suitable type of partition describedherein, such as, for example, a droplet). The oligonucleotides may bereleasably attached to the bead (e.g., releasable from the bead uponapplication of a stimulus to the bead, such as, for example, a thermalstimulus, a photo stimulus, and a chemical stimulus) as describedelsewhere herein, and/or may comprise one or more functional sequences(e.g., a primer sequence, a primer annealing sequence, an immobilizationsequence, any other suitable functional sequence described elsewhereherein, etc.) and/or one or more sequencing primer sequences asdescribed elsewhere herein. Moreover, any suitable number ofoligonucleotides may be attached to the bead, including numbers ofoligonucleotides attached to beads described elsewhere herein.

Within the partition, the oligonucleotides may be attached to fragmentsof the nucleic segment or to copies of portions of the nucleic acidsegment, such that the fragments or copies are also attached to thecommon nucleic barcode sequence. The fragments may be overlappingfragments of the nucleic acid segment and may, for example, providegreater than 2× coverage, greater than 5× coverage, greater than 10×coverage, greater than 20× coverage, greater than 40× coverage, or evengreater coverage of the nucleic acid segment. In some cases, theoligonucleotides may comprise a primer sequence capable of annealingwith a portion of the nucleic acid segment or a complement thereof. Insome cases, the oligonucleotides may be attached by extending the primersequences of the oligonucleotides to replicate at least a portion of thenucleic acid segment or complement thereof, to produce a copy of atleast a portion of the nucleic acid segment comprising theoligonucleotide, and, thus, the common nucleic acid barcode sequence.Following attachment of the oligonucleotides to the fragments of thenucleic acid segment or to the copies of the portions of the nucleicacid segment, the fragments of the nucleic acid segment or the copies ofthe portions of the nucleic acid segment and the attachedoligonucleotides (including the oligonucleotide's barcode sequence) maybe sequenced via any suitable sequencing method, including any type ofsequencing method described herein, to provide a plurality of barcodedfragment sequences or barcoded copy sequences. Following sequencing, thefragments of the nucleic acid segment or the copies of the portions ofthe nucleic acid segment can be characterized as being linked within thenucleic acid segment at least in part, upon their attachment to thecommon nucleic acid barcode sequence. As will be appreciated, suchcharacterization may include sequences that are characterized as beinglinked and contiguous, as well as sequences that may be linked withinthe same fragment, but not as contiguous sequences. Moreover, thebarcoded fragment sequences or barcoded copy sequences generated duringsequencing can be assembled into one or more contiguous nucleic acidsequences based at least in part on the common nucleic acid barcodesequence and/or a non-barcode portion of the barcoded fragment sequencesor barcoded copy sequences.

In some cases, a plurality of nucleic acid segments (e.g., fragments ofat least a portion of a genome, as described elsewhere herein) may beco-partitioned with a plurality of different beads in a plurality ofseparate partitions, such that each partition of a plurality ofdifferent partitions of the separate partitions contains a single bead.The plurality of different beads may comprise a plurality of differentbarcode sequences (e.g., at least 1,000 different barcode sequences, atleast 10,000 different barcode sequences, at least 100,000 differentbarcode sequences, at least 1,000,000 different barcodes sequences, orany other number of different barcode sequences as described elsewhereherein). In some cases, two or more, three or more, four or more, fiveor more, six or more, seven or more of the plurality of separatepartitions may comprise beads that comprise the same barcode sequence.In some cases, at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%of the separate partitions may comprise beads having the same barcodesequence. Moreover, each bead may comprise a plurality of attachedoligonucleotides that include a common nucleic acid barcode sequence.

Following co-partitioning, barcode sequences can be attached tofragments of the nucleic acid segments or to copies of portions of thenucleic acid segments in each partition. The fragments of the nucleicacid segments or the copies of the portions of the nucleic acid segmentscan then be pooled from the separate partitions. After pooling, thefragments of the nucleic acid segments or copies of the portions of thenucleic acid segments and any associated barcode sequences can besequenced (e.g., using any suitable sequencing method, including thosedescribed herein) to provide sequenced fragment or sequenced copies. Thesequenced fragments or sequenced copies can be characterized as derivingfrom a common nucleic acid segment, based at least in part upon thesequenced fragments or sequenced copies comprising a common barcodesequence. Moreover, sequences obtained from the sequenced fragments orsequenced copies may be assembled to provide a contiguous sequence of asequence (e.g., at least a portion of a genome) from which the sequencedfragments or sequenced copies originated. Sequence assembly from thesequenced fragments or sequenced copies may be completed based, at leastin part, upon each of a nucleotide sequence of the sequenced fragmentsand a common barcode sequence of the sequenced fragments.

In another example method, a target nucleic acid may be characterized bypartitioning fragments of the target nucleic acid into a plurality ofdroplets. Each droplet can comprise a bead attached to a plurality ofoligonucleotides comprising a common barcode sequence. The commonbarcode sequence can be attached to fragments of the fragments of thetarget nucleic acid in the droplets. The droplets can then be pooled andthe fragments and associated barcode sequences of the pooled dropletssequenced using any suitable sequencing method, including sequencingmethods described herein. Following sequencing, the fragments of thefragments of the target nucleic acid may be mapped to the fragments ofthe target nucleic acid based, at least in part, upon the fragments ofthe fragments of the target nucleic acid comprising a common barcodesequence.

The application of the methods, compositions and systems describedherein in sequencing may generally be applicable to any of a variety ofdifferent sequencing technologies, including NGS sequencing technologiessuch as Illumina MiSeq, HiSeq and X10 Sequencing systems, as well assequencing systems available from Life Technologies, Inc., such as theIon Torrent line of sequencing systems. While discussed in terms ofbarcode sequences, it will be appreciated that the sequenced barcodesequences may not include the entire barcode sequence that is included,e.g., accounting for sequencing errors. As such, when referring tocharacterization of two barcode sequences as being the same barcodesequence, it will be appreciated that this may be based upon recognitionof a substantial portion of a barcode sequence, e.g., varying by fewerthan 5, 4, 3, 2 or even a single base.

Sequencing from Small Numbers of Cells

Methods provided herein may also be used to prepare polynucleotidescontained within cells in a manner that enables cell-specificinformation to be obtained. The methods enable detection of geneticvariations from very small samples, such as from samples comprisingabout 10-100 cells. In some cases, about 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90 or 100 cells may be used in the methods described herein. Insome cases, at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or100 cells may be used in the methods described herein. In other cases,at most about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may beused in the methods described herein.

In an example, a method may comprise partitioning a cellular sample (orcrude cell extract) such that at most one cell (or extract of one cell)is present within a partition, e.g., fluidic droplet, and isco-partitioned with the barcode oligonucleotides, e.g., as describedabove. Processing then involves lysing the cells, fragmenting thepolynucleotides contained within the cells, attaching the fragmentedpolynucleotides to barcoded beads, pooling the barcoded beads, andsequencing the resulting barcoded nucleic acid fragments.

As described elsewhere herein, the barcodes and other reagents may beencapsulated within, coated on, associated with, or dispersed within abead (e.g. gel bead). The bead may be loaded into a fluidic dropletcontemporaneously with loading of a sample (e.g. a cell), such that eachcell is contacted with a different bead. This technique may be used toattach a unique barcode to oligonucleotides obtained from each cell. Theresulting tagged oligonucleotides may then be pooled and sequenced, andthe barcodes may be used to trace the origin of the oligonucleotides.For example, oligonucleotides with identical barcodes may be determinedto originate from the same cell, while oligonucleotides with differentbarcodes may be determined to originate from different cells.

The methods described herein may be used to detect a specific genemutation that may indicate the presence of a disease, such as cancer.For example, detecting the presence of a V600 mutation in the BRAF geneof a colon tissue sample may indicate the presence of colon cancer. Inother cases, prognostic applications may include the detection of amutation in a specific gene or genes that may serve as increased riskfactors for developing a specific disease. For example, detecting thepresence of a BRCA1 mutation in a mammary tissue sample may indicate ahigher level of risk to developing breast cancer than a person withoutthis mutation. In some examples, this disclosure provides methods ofidentifying mutations in two different oncogenes (e.g., KRAS and EGRF).If the same cell comprises genes with both mutations, this may indicatea more aggressive form of cancer. In contrast, if the mutations arelocated in two different cells, this may indicate that the cancer may bemore benign, or less advanced.

Analysis of Gene Expression

Methods of the disclosure may be applicable to processing samples forthe detection of changes in gene expression. A sample may comprise acell, mRNA, or cDNA reverse transcribed from mRNA. The sample may be apooled sample, comprising extracts from several different cells ortissues, or a sample comprising extracts from a single cell or tissue.

Cells may be placed directly into a fluidic droplet and lysed. Afterlysis, the methods of the disclosure may be used to fragment and barcodethe oligonucleotides of the cell for sequencing. Oligonucleotides mayalso be extracted from cells prior to introducing them into a fluidicdroplet used in a method of the disclosure. Reverse transcription ofmRNA may be performed in a fluidic droplet described herein, or outsideof such a fluidic droplet. Sequencing cDNA may provide an indication ofthe abundance of a particular transcript in a particular cell over time,or after exposure to a particular condition.

Partitioning Polynucleotides from Cells or Proteins

In one example the compositions, methods, devices, and kits provided inthis disclosure may be used to encapsulate cells or proteins within thefluidic droplets. In one example, a single cell or a plurality of cells(e.g., 2, 10, 50, 100, 1000, 10000, 25000, 50000, 10000, 50000, 1000000,or more cells) may be loaded onto, into, or within a bead along with alysis buffer within a fluidic droplet and incubated for a specifiedperiod of time. The bead may be porous, to allow washing of the contentsof the bead, and introduction of reagents into the bead, whilemaintaining the polynucleotides of the one or more cells (e.g.chromosomes) within the fluidic droplets. The encapsulatedpolynucleotides of the one or more cells (e.g. chromosomes) may then beprocessed according to any of the methods provided in this disclosure,or known in the art. This method can also be applied to any othercellular component, such as proteins.

Epigenetic Applications

Compositions, methods, devices, and kits of this disclosure may beuseful in epigenetic applications. For example, DNA methylation can bein indicator of epigenetic inheritance, including single nucleotidepolymorphisms (SNPs). Accordingly, samples comprising nucleic acid maybe treated in order to determine bases that are methylated duringsequencing. In some cases, a sample comprising nucleic acid to bebarcoded may be split into two aliquots. One aliquot of the sample maybe treated with bisulfite in order to convert unmethylated cytosinecontaining nucleotides to uracil containing nucleotides. In some cases,bisulfite treatment can occur prior to sample partitioning or may occurafter sample partitioning. Each aliquot may then be partitioned (if notalready partitioned), barcoded in the partitions, and additionalsequences added in bulk as described herein to generate sequencer-readyproducts. Comparison of sequencing data obtained for each aliquot (e.g.,bisulfite-treated sample vs. untreated sample) can be used to determinewhich bases in the sample nucleic acid are methylated.

In some cases, one aliquot of a split sample may be treated withmethylation-sensitive restriction enzymes (MSREs). Methylation specificenzymes can process sample nucleic acid such that the sample nucleicacid is cleaved as methylation sites. Treatment of the sample aliquotcan occur prior to sample partitioning or may occur after samplepartitioning and each aliquot may be partitioned used to generatebarcoded, sequencer-ready products. Comparison of sequencing dataobtained for each aliquot (e.g., MSRE-treated sample vs. untreatedsample) can be used to determine which bases in the sample nucleic acidare methylated.

Low Input DNA Applications

Compositions and methods described herein may be useful in the analysisand sequencing of low polynucleotide input applications. Methodsdescribed herein, such as PHASE, may aid in obtaining good data qualityin low polynucleotide input applications and/or aid in filtering outamplification errors. These low input DNA applications include theanalysis of samples to sequence and identify a particular nucleic acidsequence of interest in a mixture of irrelevant or less relevant nucleicacids in which the sequence of interest is only a minority component, tobe able to individually sequence and identify multiple different nucleicacids that are present in an aggregation of different nucleic acids, aswell as analyses in which the sheer amount of input DNA is extremelylow. Specific examples include the sequencing and identification ofsomatic mutations from tissue samples, or from circulating cells, wherethe vast majority of the sample will be contributed by normal healthycells, while a small minority may derive from tumor or other cancercells. Other examples include the characterization of multipleindividual population components, e.g., in microbiome analysisapplications, where the contributions of individual population membersmay not otherwise be readily identified amidst a large and diversepopulation of microbial elements. In a further example, being able toindividually sequence and identify different strands of the same regionfrom different chromosomes, e.g., maternal and paternal chromosomes,allows for the identification of unique variants on each chromosome.Additional examples of low polynucleotide input applications of thecompositions, methods, and systems described herein are set forth inU.S. Provisional Patent Application No. 62/017,580 (Attorney Docket No.43487-727.101), filed of even date herewith.

The advantages of the methods and systems described herein are clearerupon a discussion of the problems confronted in the present state of theart. In analyzing the genetic makeup of sample materials, e.g., cell ortissue samples, most sequencing technologies rely upon the broadamplification of target nucleic acids in a sample in order to createenough material for the sequencing process. Unfortunately, during theseamplification processes, majority present materials will preferentiallyoverwhelm portions of the samples that are present at lower levels. Forexample, where a genetic material from a sample is comprised of 95%normal tissue DNA, and 5% of DNA from tumor cells, typical amplificationprocesses, e.g., PCR based amplification, will quickly amplify themajority present material to the exclusion of the minority presentmaterial. Furthermore, because these amplification reactions aretypically carried out in a pooled context, the origin of an amplifiedsequence, in terms of the specific chromosome, polynucleotide ororganism will typically not be preserved during the process.

In contrast, the methods and systems described herein partitionindividual or small numbers of nucleic acids into separate reactionvolumes, e.g., in droplets, in which those nucleic acid components maybe initially amplified. During this initial amplification, a uniqueidentifier may be coupled to the components to the components that arein those separate reaction volumes. Separate, partitioned amplificationof the different components, as well as application of a uniqueidentifier, e.g., a barcode sequence, allows for the preservation of thecontributions of each sample component, as well as attribution of itsorigin, through the sequencing process, including subsequentamplification processes, e.g., PCR amplification.

Non-Overlapping Fragmentation

This disclosure provides methods, compositions, systems, and devices forprocessing polynucleotides. In one example, a method provided hereincomprises: (a) providing a target polynucleotide; (b) fragmenting thetarget polynucleotide to generate a plurality of non-overlapping firstpolynucleotide fragments; (c) partitioning the first polynucleotidefragments to generate partitioned first polynucleotide fragments,wherein at least one partition of the partitioned first polynucleotidefragments comprises a first polynucleotide fragment with a uniquesequence within the at least one partition; and (d) fragmenting thepartitioned first polynucleotide fragments, to generate a plurality ofnon-overlapping second polynucleotide fragments.

In some of the methods provided in this disclosure, a third and fourthset of polynucleotide fragments are generated by performing the methoddescribed above and additionally performing a method comprising: (a)fragmenting the target polynucleotide to generate a plurality ofnon-overlapping third polynucleotide fragments; (b) partitioning thethird polynucleotide fragments to generate partitioned thirdpolynucleotide fragments, wherein at least one partition of thepartitioned third polynucleotide fragments comprises a thirdpolynucleotide fragment with a unique sequence within the at least onepartition; and (c) fragmenting the partitioned third polynucleotidefragments to generate a plurality of non-overlapping fourthpolynucleotide fragments.

The third polynucleotide fragments may overlap with the firstpolynucleotide fragments. The fourth polynucleotide fragments mayoverlap with the second polynucleotide fragments.

The target polynucleotide may be, for example, DNA, RNA, cDNA, or anyother polynucleotide.

In some cases, at least one of the first, second, third, and fourthpolynucleotide fragments are generated by an enzyme. The enzyme may be arestriction enzyme. The restriction enzyme used to generate the firstpolynucleotide fragments may be different from the restriction enzymeused to generate the third polynucleotide fragments. The restrictionenzyme used to generate the second polynucleotide fragments may bedifferent from the restriction enzyme used to generate the fourthpolynucleotide fragments. The restriction enzymes may have a recognitionsite of at least about six nucleotides in length.

The fragments can be of a variety of lengths. For example, the firstand/or third polynucleotide fragments may have a median length of leastabout 10,000 nucleotides. The second or fourth polynucleotide fragmentsmay have a median length of less than about 200 nucleotides.

The fragments can be attached to barcodes. For example, the secondpolynucleotide fragments and/or the fourth polynucleotide fragments maybe attached to barcodes, to generate barcoded second and/or fourthpolynucleotide fragments. The barcodes may be polynucleotide barcodes.The attachment of the barcodes to the polynucleotide fragments may beperformed using an enzyme. The enzyme may be a ligase. The barcodedfragments may be pooled. Unpooled or pooled barcoded fragments may besequenced.

In some cases, one or more steps of the methods described in thisdisclosure may be performed within a device. The device may comprise atleast one well. The well may be a microwell. Any of the partitioningsteps described in this disclosure may be performed by dispensing into amicrowell.

The microwell (or well) may comprise reagents. These reagents may be anyreagent, including, for example, barcodes, enzymes, adapters, andcombinations thereof. The reagents may be physically separated from apolynucleotide sample placed in the microwell. This physical separationmay be accomplished by containing the reagents within a microcapsulethat is placed within a microwell. The physical separation may also beaccomplished by dispensing the reagents in the microwell and overlayingthe reagents with a layer that is, for example, dissolvable, meltable,or permeable prior to introducing the polynucleotide sample into themicrowell. This layer may be, for example, an oil, wax, membrane, or thelike. The microwell may be sealed at any point, for example afteraddition of the microcapsule, after addition of the reagents, or afteraddition of either of these components plus a polynucleotide sample.

Partitioning may also be performed by a variety of other means,including through the use of fluid flow in microfluidic channels, byemulsification, using spotted arrays, by surface acoustic waves, and bypiezoelectric droplet generation.

Additional methods of fragmenting nucleic acids that are compatible withthe methods provided herein include mechanical disruption, sonication,chemical fragmentation, treatment with UV light, and heating, andcombinations thereof. These methods may be used to fragment, forexample, the partitioned first or third polynucleotide fragmentsdescribed above.

Partitioning may be done at any time. For example, the firstpolynucleotide fragments and/or the third polynucleotide fragments mayeach be further partitioned into two or more partitions before furtherprocessing.

Pseudo-Random Fragmentation

This disclosure provides methods for pseudo-random fragmentation ofpolynucleotides. In some cases, such methods comprise: (a) providing atarget polynucleotide; (b) fragmenting the target polynucleotide togenerate a plurality of first polynucleotide fragments; (c) partitioningthe first polynucleotide fragments to generate partitioned firstpolynucleotide fragments, such that at least one partition comprises afirst polynucleotide fragment with a unique sequence within the at leastone partition; and (d) fragmenting the partitioned first polynucleotidefragments with at least one restriction enzyme in at least onepartition, to generate a plurality of second polynucleotide fragments,wherein the partitioned first polynucleotide fragment is fragmented withat least two restriction enzymes across all partitions.

In some cases, at least two restriction enzymes are disposed within thesame partition. In some cases, at least two restriction enzymes aredisposed across a plurality of different partitions.

The pseudo-random fragmentation methods can be performed in order toyield fragments of a certain size. In some cases, at least about 50% ofthe nucleotides within a target polynucleotide are within about 100nucleotides of a restriction site of a restriction enzyme used toperform pseudo-random fragmentation. In some cases, at most about 25% ofthe nucleotides within a target polynucleotide are within about 50nucleotides of a restriction site of a restriction enzyme used toperform pseudo-random fragmentation. In some cases, at most about 10% ofthe nucleotides within a target polynucleotide are more than about 200nucleotides from a restriction site a restriction enzyme used to performpseudo-random fragmentation.

A polynucleotide may be treated with two or more restriction enzymesconcurrently or sequentially.

The pseudo-randomly fragmented polynucleotides may be attached tobarcodes, to generate barcoded polynucleotide fragments. The barcodedpolynucleotides may be pooled and sequenced.

The number of partitions holding the partitioned first polynucleotidefragments may be at least about 1,000 partitions. The volume of thesepartitions may be less than about 500 nanoliters.

Each enzyme may occupy an equivalent number of partitions, or eachenzyme may occupy a different number of partitions.

Restriction Enzyme-Mediated Recycling

This disclosure provides methods for recycling certain unwanted reactionside products back into starting materials that can be used to generatea desired product. In some cases, these methods comprise: (a) providinga first polynucleotide, a second polynucleotide, a first restrictionenzyme, and a second restriction enzyme, wherein the firstpolynucleotide comprises a target polynucleotide or a fragment thereof;and (b) attaching the first polynucleotide to the second polynucleotide,to generate a polynucleotide product, wherein the first restrictionenzyme cuts a polynucleotide generated by attachment of the firstpolynucleotide to itself, the second restriction enzyme cuts apolynucleotide generated by attachment of the second polynucleotide toitself, and neither the first restriction enzyme nor the secondrestriction enzyme cuts the polynucleotide product.

The first polynucleotide may be generated in the same reaction volume asthe polynucleotide product, or in a different reaction volume. Thetarget polynucleotide may be, for example, a fragment of genomic DNA.

The second polynucleotide may be generated in the same reaction volumeas the polynucleotide product, or in a different reaction volume. Thesecond polynucleotide may be, for example, a barcode or an adapter.

The first restriction enzyme may have a recognition site of at mostabout four nucleotides in length. The second restriction enzyme may havea recognition site of at least about six nucleotides in length. Thefirst restriction enzyme may have a recognition site of about fournucleotides in length. The second restriction enzyme may have arecognition site of at least about five nucleotides in length.

The first and second restriction enzymes may generate ligationcompatible ends. These ends may have single-stranded overhangs (i.e.,“sticky ends”) or be blunt. The sticky ends may match in sequence andorientation, to allow ligation. The attachment step may be performed byligation.

The sequence 5′ to the ligation compatible end generated by the firstrestriction enzyme may be different from the sequence 5′ to the ligationcompatible end generated by the second restriction enzyme. This willensure that the desired product cannot be re-cut by either restrictionenzyme.

The sequence 3′ to the ligation compatible end generated by the firstrestriction enzyme may be different from the sequence 3′ to the ligationcompatible end generated by the second restriction enzyme. This willensure that the desired product cannot be re-cut by either restrictionenzyme. Given the criteria provided throughout this specification, oneof ordinary skill in the art will recognize that many pairs of enzymesare suitable for use with this method.

The recycling may provide increased yield of the desired product, forexample at least about 75% (w/w).

Also provided by this disclosure is a polynucleotide fragment generatedby any of the methods provided herein, devices for performing themethods provided herein, and systems for performing the methods providedherein.

The methods provided in this disclosure (and portions thereof) may alsobe used with each other. For example, the non-overlapping fragmentationmethods may be used alone and/or with the pseudo-random fragmentationmethods and/or with the restriction enzyme-mediated recycling methods.Likewise, the pseudo-random fragmentation methods may be used aloneand/or with the non-overlapping fragmentation methods and/or with therestriction enzyme-mediated recycling methods. Similarly, therestriction enzyme-mediated recycling methods may be used alone and/orwith the non-overlapping fragmentation methods and/or with thepseudo-random fragmentation methods.

The term “about,” as used herein and throughout the disclosure,generally refers to a range that may be 15% greater than or 15% lessthan the stated numerical value within the context of the particularusage. For example, “about 10” would include a range from 8.5 to 11.5.

As will be appreciated, the instant disclosure provides for the use ofany of the compositions, libraries, methods, devices, and kits describedherein for a particular use or purpose, including the variousapplications, uses, and purposes described herein. For example, thedisclosure provides for the use of the compositions, methods, libraries,devices, and kits described herein in partitioning species, inpartitioning oligonucleotides, in stimulus-selective release of speciesfrom partitions, in performing reactions (e.g., ligation andamplification reactions) in partitions, in performing nucleic acidsynthesis reactions, in barcoding nucleic acid, in preparingpolynucleotides for sequencing, in sequencing polynucleotides, inpolynucleotide phasing (see e.g., U.S. Provisional Patent ApplicationNo. 62/017,808 (Attorney Docket No. 43487-726.101) filed of even dateherewith), in sequencing polynucleotides from small numbers of cells, inanalyzing gene expression, in partitioning polynucleotides from cells,in mutation detection, in neurologic disorder diagnostics, in diabetesdiagnostics, in fetal aneuploidy diagnostics, in cancer mutationdetection and forensics, in disease detection, in medical diagnostics,in low input nucleic acid applications, such as circulating tumor cell(CTC) sequencing, in a combination thereof, and in any otherapplication, method, process or use described herein.

Any concentration values provided herein are provided as admixtureconcentration values, without regard to any in situ conversion,modification, reaction, sequestration or the like. Moreover, whereappropriate, the sensitivity and/or specificity of methods (e.g.,sequencing methods, barcoding methods, amplification methods, targetedamplification methods, methods of analyzing barcoded samples, etc.)described herein may vary. For example, a method described herein mayhave specificity of greater than 50%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% and/or asensitivity of greater than 50%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.

X. EXAMPLES Example 1: Creation of Gel Beads Functionalized withAcrydite Primer

Gel beads are produced according to the method illustrated in FIG. 2. Innuclease free water, 1 mL stock solutions are prepared at the followingconcentrations: an acrylamide precursor (Compound A)=40% (v/v) stocksolution, a crosslinker (Bis-acryloyl cystamine—Compound B)=3.19 mg/mLin 50:50 mix of acetonitrile:water, an initiator (Compound C)=20 mg/mL,and di-sulfide acrydite primer (Compound D)=1 mM. From these stocksolutions, 1 mL of an aqueous Gel Bead (GB) working solution is preparedby mixing the following volumes: nuclease free water=648 μL, CompoundA=150 μL, Compound B=100 μL, Compound C=100 μL, and Compound D=2 μL.Stock solutions of Compound A and B and GB working solutions areprepared daily.

The Gel Bead (GB) working solution, 201, is an aqueous fluid thatcontains the crosslinker, BAC, and a polymer precursor solution withdi-sulfide-modified acrydite oligonucleotides at a concentration ofbetween about 0.1 and about 100 μm. The second fluid, 202, is afluorinated oil containing the surfactant, Krytox FSH 1.8% w/w HFE 7500.The accelerator, tetramethylethylenediamine (TEMED) is added a) to theoil prior to droplet generation, 203, b) in the line after dropletgeneration, 205, and/or c) to the outlet reservoir after dropletgeneration, 206 to give a final concentration of 1% (v/v). TEMED is madefresh daily. Gel beads are generated by sending the aqueous and oilphase fluids to a droplet generator, 204. Polymerization is initiatedimmediately after droplet generation and continues to the outlet well.Gelation is considered complete after 15-20 minutes. After gelation,generated gel beads are subjected to continuous phase exchange bywashing in HFE 7500, 207, to remove excess oil, and re-suspending thebeads in aqueous solution. In some cases, the resulting beads may bepresent in an agglomeration. The agglomeration of gel beads areseparated into individual gel beads with vortexing. Gel beads arevisualized under a microscope.

Example 2: Creation of Barcoded Gel Beads by Limiting Dilution

Functionalized gel beads are produced by limiting dilution according tothe method illustrated in FIG. 3A and FIGS. 4A-4N. Gel beads withacrydite oligonucleotides (with or without a di-sulfide modification),301, 401, are mixed with barcode-containing template sequences, 302, ata limiting dilution. PCR reagents, 303, including a biotin labeled readprimer, 406, are mixed with the gel beads and template sequences, 304.The beads, barcode template, and PCR reagents are emulsified into agel-water-oil emulsion by shaking/agitation, flow focusing, ormicrosieve, 305, preferably such that at most one barcode template ispresent in a partition (e.g., droplet) within the emulsion. The emulsionis exposed to one or more thermal cycles, 306. The first thermocycleincorporates the complement barcode sequence, 408, and immobilizes itonto the gel bead.

Continued thermal cycling is performed to clonally amplify the barcodethroughout the gel bead and to incorporate the 5′ biotin labeled primerinto the complementary strand for downstream sorting of beads whichcontain barcode sequences from those that do not. The emulsion isbroken, 307, by adding perfluorodecanol, removing the oil, washing withHFE-7500, adding aqueous buffer, centrifuging, removing supernatant,removing undesired products (e.g. primer dimers, starting materials,deoxynucleotide triphosphates (dNTPs), enzymes, etc.) and recoveringdegradable gel beads into an aqueous suspension. The functionalized gelbeads are re-suspended in high salt buffer, 308. Streptavidin-labeledmagnetic beads are added to the re-suspension, which is then incubatedto allow binding to gel beads attached to biotinylated barcodes 308,410. A magnetic device is then used to separate positive barcoded gelbeads from beads that are not attached to barcode, 308. Denaturationconditions, 309, (e.g. heat or chemical denaturant) are applied to thegel beads in order to separate the biotinylated complementary strandfrom the barcoded beads. The magnetic beads are subsequently removedfrom the solution; and the resulting solution ofpartially-functionalized barcoded beads is pooled for furtherprocessing.

Example 3: Further Functionalization of Barcoded Beads

As shown in FIG. 3B, the barcoded gel beads, 311, from Example 2, arefurther functionalized as follows. The beads are combined with anadditional template oligonucleotide, 310, (such as an oligonucleotidecontaining a random N-mer sequence, 413, as shown in FIGS. 4I and 4J),and PCR reagents, 312, 313, and subjected to conditions to enablehybridization of the template oligonucleotide with the read primerattached to the gel bead. An extension reaction is performed so that thebarcode strands are extended, 314, thereby incorporating thecomplementary sequence of the template oligonucleotide. Resultingfunctionalized gel beads are re-suspended in aqueous buffer, 315, andexposed to heating conditions to remove complement strands, 316, andplaced into aqueous storage buffer, 317.

Example 4: Step-by-Step Description of Bead Functionalization

FIGS. 4A-4N provides a step-by-step description of an example process offunctionalizing the gel beads with barcodes and random N-mers. As shownin FIG. 4A, the process begins with gel beads, 401, that are attached toa universal primer, such as a P5 primer (or its complement), 403. Thebeads may be linked to the primer via a di-sulfide bond, 402. The gelbeads are provided in an aqueous solution (g/w). Using a limitingdilution and partitioning, unique barcode sequence templates, 405, arecombined with the beads such that at most one unique barcode sequenceoccupies the same partition as a gel bead. Generally, the partitions areaqueous droplets within a gel/water/oil (g/w/o) emulsion. As shown inFIG. 4B, the barcode sequence template, 405, is contained within alarger nucleotide strand that contains a sequence, 404, that iscomplementary to the universal primer 403, as well as a sequence, 407,that is identical in sequence to a biotin labeled read primer, 406.

As shown in FIG. 4C, an amplification reaction is then conducted toincorporate the complement, 408, of the barcode template, 405, onto thestrand that is attached to the bead. The amplification reaction alsoresults in incorporation of a sequence, 415, that is complementary tosequence, 407. Additional amplification cycles result in hybridizationof the biotin labeled read primer, 406, to sequence, 415 (FIG. 4D), andthe biotin labeled read primer is then extended (FIG. 4E). The emulsionmay then be broken, and the gel beads may then be pooled into agel/water common solution.

In the gel/water solution, magnetic capture beads, 409, are then used tocapture the biotinylated nucleic acids attached to the gel beads, whichare then isolated from beads that only contain the original primer(FIGS. 4F and 4G). The biotinylated strand is then removed from thestrand attached to the gel bead (FIG. 4H). Random N-mer sequences, 414,may then be attached to the strands attached to the gel bead. For eachgel bead, an identical barcode sequence, 408, is attached to each primerthroughout the gel bead; each barcode sequence is then functionalizedwith a random N-mer sequence, 414, such that multiple different randomN-mer sequences are attached to each bead. For this process, a randomN-mer template sequence, 413, linked to a sequence, 412, complementaryto sequence, 415, is introduced to the solution containing the pooledbeads (FIG. 4I). The solution is subjected to conditions to enablehybridization of the template to the strand attached to the bead andsequence 415 is extended to include the random N-mer, 414. (FIG. 4J).The fully functionalized beads (FIG. 4K) are then combined with a samplenucleic acid and a reducing agent (e.g., dithiothreitol (DTT) at aconcentration of 1 mM) and partitioned within droplets of agel/water/oil emulsion (FIG. 4L). This combining step may be conductedwith a microfluidic device (FIG. 5A). The gel beads are then degradedwithin each partition (e.g., droplet) such as by the action of areducing agent, and the barcoded sequence is released from the droplet(FIGS. 4M and 4N). The random N-mer within the barcoded sequence mayserve as a primer for amplification of the sample nucleic acid.

Example 5: Use of a Microfluidic Chip to Combine the Gel-Beads-inEmulsions (GEMs) with Sample

The functionalized gel beads may be combined with sample using adouble-cross microfluidic device illustrated in FIG. 5B. Degradable gelbeads are introduced to the fluidic input, 501, in a fluid stream, whichcontains about 7% glycerol. The experimental sample of interest isintroduced to the fluidic input, 502, in a fluid stream, which isaqueous phase. The reducing agent, dithiothreitol (DTT) at aconcentration of about 1 mM is introduced to the fluidic input, 503, ina fluid stream, which contains about 7% glycerol. Fluidic inputs 501,502, and 503 mix at a microfluidic cross junction, 504, and enter asecond microfluidic cross junction, 506. The second microfluidic crossjunction can be used to produce emulsified (w/o) droplets containing theel beads. Fluidic input, 505, is used to introduce oil with 2% (w/w) biskrytox peg (BKP). Individual droplets exiting from the secondmicrofluidic cross junction, 507, are added into microplate wells, FIG.5C, for further downstream applications. FIG. 5D is an image of dropletsgenerated in the absence of DTT (and therefore containing gel beads).FIG. 5E is an image of droplets generated with DTT that caused theinternal gel beads to degrade.

Example 6: Fluorescent Identification of Positive Gel Beads

FIGS. 6A-6F depicts images of gel beads containing amplified nucleicacids that have been labeled with a fluorescent label. Functionalizationof the gel beads is first performed using a limiting dilution so thatonly a portion of the gel beads are functionalized with barcodes. Gelbeads suspended in a bis krytox peg (BKP) emulsion are imaged at 4×magnification following PCR thermocycling but before washing. The brightfield image, FIG. 6A, shows all emulsion-generated droplets, and thefluorescent image, FIG. 6B, shows only positive functionalized gelbeads. Many non-fluorescent droplets are generated indicating emptydroplets, which do not contain either gel bead and/or oligonucleotide.Empty droplets are washed away by multiple re-suspensions and washing inHFE-7500. FIGS. 6C and 6D show positive gel bead enrichment followingemulsion breaking and further wash steps. The bright field images (4×),FIG. 6C, and (10×) FIG. 6E, show all gel beads. The fluorescent images(4×), FIG. 6D, and (10×), FIG. 6F, show 30% positive beads from SYBRstaining. The 30% positive bead result matches predicted value from gDNAinput.

FIGS. 7A-7C shows images of gel beads containing single stranded (ss)DNA, double-stranded (ds) DNA, and denatured, ssDNA. Gel beads stainedwith 1× EvaGreen are brighter in the presence of dsDNA as evident fromthe fluorescent images taken at step 1: Make (ssDNA), FIG. 7A, step 2:Extension (dsDNA), FIG. 7B, and step 3: Denature (ssDNA), FIG. 7C.Fluorescent images show that beads become brighter after extension andbecome dimmer after denaturation.

Example 7: Enrichment of Positive Gel Beads Using Streptavidin-CoatedMagnetic Beads

Enrichment of positive gel beads using streptavidin-coated magneticbeads is depicted in FIGS. 8A-8F. FIG. 8A (bright field) and FIG. 8B(fluorescent) provides images of SYBR-stained gel beads 24 hoursfollowing the addition of magnetic beads. Magnetic coated positive gelbeads are brighter due to SYBR staining. Bright field images before,FIG. 8C, and after sorting, FIG. 8D, at a magnetic bead concentration of40 mg/mL, show positive gel bead enrichment, where coated beads areoptically brighter. Bright field images before, FIG. 8E, and aftersorting, FIG. 8F, at a magnetic bead concentration of 60 mg/mL, showpositive gel bead enrichment, where coated beads are optically brighter.At each magnetic bead working concentration, a single gel bead is coatedby about 100-1000 magnetic beads.

Example 8: Dissolution of Gel Beads

Heating gel beads in basic solution degrades the gel beads as evident inFIGS. 9A-9D. Gel beads are heated in basic solution at 95° C. andmonitored at 5 minute heating intervals: t=0 min, FIG. 9A, t=5 min, FIG.9B, t=10 min, FIG. 9C, t=15 min, FIG. 9D. Following 15 minutes, gelbeads are completely degraded. Gel beads more than double in size whilethey are degrading. FIGS. 10A-10G depicts dissolution of the gel beadsusing tris(2-carboxyethyl)phosphine (TCEP), which is an effective andirreversible di-sulfide bond reducing agent. Functionalized gel beads,FIG. 10A, are placed into basic solution, pH=8, with 1 mM TCEP andmonitored at 2 minute intervals: t=0 min, FIG. 10B, t=2 min, FIG. 10C,t=4 min, FIG. 10D, t=6 min, FIG. 10E, t=8 min, FIG. 10F, and t=10 min,FIG. 10G. Between about 6 and about 10 minutes, the functionalized gelbeads are completely degraded.

Example 9: Analysis of Content After Dissolution Gel Beads (GB)

An analysis of content attached to gel beads is provided in FIGS.11A-11D, and FIG. 12. Gel beads are functionalized, 1101, with barcodeor barcode complement (N12C) and a random N-mer (8mer) that is 8nucleotides in length, 1102. The random N-mer is attached by performinga primer extension reaction using a template construct containing R1Cand a random N-mer 1102. The length of the entire oligonucleotide strand(including the bar code and random N-mer) is 82 bp, 1101. The strandlength of the random N-mer and the R1C is 42 base pairs (bp), 1102. Theextension reaction is performed using a KAPA HIFI RM Master Mix underhigh primer concentration (10 μm) at 65° C. for one hour. Increasing thenumber of wash steps before the step of degrading the gel beads resultsin a reduction in the amount of primer dimers within the sample. When nowashes are performed, 1103, both 42 bp products, 1106, and 80 bpproducts, 1107, can be observed. After three washes, the level of primerdimer, 1104, is reduced relative to the no-wash experiment. After sixwashes, 1105, 80 bp products, 1107, are observed, but no primer dimersare observed.

The six-wash experiment can also be performed using six differenttemperatures (65° C., 67° C., 69° C., 71° C., 73° C., 75° C., FIG. 11C)for the extension step. In this specific example, a high primerconcentration (10 μm) is used and the extension step lasts one hour. Itappears that 67° C. is the optimal temperature for both optimizing thelevel of 80 bp products and minimizing the number of 42 bp products,1109.

The temperature, 67° C., is chosen for subsequent denaturation studies.Heat denaturation of the complementary strand, wherein the sample isheated to 95° C. six times and washed to remove complementary strand,results in an 84 bp peak, 1202, before denaturation, and shows a reducedpeak, 1201, following denaturation. The control value measured from step1 is shown at 1203.

Example 10: Creation of Barcoded Gel Beads by Partitioning in Wells

Functionalized beads are produced by partitioning in wells according tothe method illustrated in FIGS. 13A and 13B. The first functionalizationstep is outlined in FIG. 13A, the second functionalization step isoutlined in FIG. 13B. An example multiplex adaptor creation scheme isoutlined in FIG. 13C and described in Example 11. As shown in FIG. 13A,functionalized beads, 1301 (e.g., beads with acrydite oligos and primer(e.g., 5′-AAUGAUACGGCGACCACCGAGA-3′ (SEQ ID NO: 4)), the template withbarcode sequence, 1302 (e.g., 5′-XXXXXXTCTCGGTGGTCGCCGTATCATT-3′ (SEQ IDNO: 5)), and appropriate PCR reagents, 1303, are mixed together,1304/1305 and divided into 384 wells of a multi-well plate. Each wellcomprises multiple copies of a unique barcode sequence and multiplebeads. Thermocycling, 1306, with an extension reaction is performed ineach individual well to form beads with attached barcodes. All wells arepooled together and cleaned up in bulk, 1307/1308.

To add a random N-mer, the partially functionalized beads, 1310, thetemplate random N-mer oligonucleotides, 1309, and the appropriate PCRreagents, 1311, are mixed together, 1312, and the functionalized beads1310 subjected to extension reactions 1313 to add a random N-mersequence complementary to the random N-mer template, to the beads.Following thermal cycling, the beads are cleaned up in bulk, 1314-1316.

Example 11: Combinatorial Plate Technique

As shown in FIG. 13C, beads 1317 attached to primers (e.g., P5oligomers, 5′-AAUGAUACGGCGACCACCGAGA-3′ (SEQ ID NO: 4)) 1318 arepartitioned into wells of a multi-well plate (such as a 5×-1 384-wellplate 1319) with multiple copies of a template 1321 comprising a uniquetemplate partial barcode sequence (e.g.,5′-XXXXXXTCTCGGTGGTCGCCGTATCATT-3 (SEQ ID NO: 5)). Extension reactions(e.g., extension of primer 1318 via template 1321) are performed togenerate Bead-PS-[5×-1], 1320 comprising an extension product (e.g., anoligonucleotide comprising primer 1318 and a partial barcode sequencecomplementary to the template partial barcode sequence) in each well.The beads are removed from the wells are pooled together and a clean-upstep is performed in bulk.

The pooled mixture is then re-divided into wells of a second multiwellplate such as a 384-well plate with 5λ-2, 1322, with each well alsocomprising an oligonucleotide comprising a second unique partial barcodesequence and a random N-mer (e.g.,5′P-YYYYYYCGCACACUCUUUCCCUACACGACGCUCUUCCGAUCUN-BLOCK (SEQ ID NO: 6)).The oligonucleotide may have a blocker oligonucleotide attached (e.g.,via hybridization) (e.g., “BLOCK”). Single-stranded ligation reactions1324 are performed between the extension product bound to the bead andthe oligonucleotide comprising the second partial barcode sequence andrandom N-mer. Following the ligation reaction, beads comprising a fullbarcode sequence (e.g., XXXXX and a random N-mer are generated, 1323(e.g., Bead-P5-[5×-1][5×-2]R1[8N-Blocker]). The beads also comprise theblocker oligonucleotide. All wells are then pooled together, theblocking groups are cleaved, and the bead products are cleaned up inbulk. Beads comprising a large diversity of barcode sequences areobtained.

Example 12: Partial Hairpin Amplification for Sequencing (PHASE)Reaction

Partial Hairpin Amplification for Sequencing (PHASE) reaction is atechnique that can be used to mitigate undesirable amplificationproducts according to the method outlined in FIGS. 14A-14C and FIGS.15A-15G by forming partial hairpin structures. Specifically, randomprimers, of about 8N-12N in length, 1404, tagged with a universalsequence portion, 1401/1402/1403, may be used to randomly prime andextend from a nucleic acid, such as, genomic DNA (gDNA). The universalsequence comprises: (1) sequences for compatibility with a sequencingdevice, such as, a flow cell (e.g. Illumina's P5, 1401, and Read 1Primer sites, 1402) and (2) a barcode (BC), 1403, (e.g., 6 basesequences). In order to mitigate undesirable consequences of such a longuniversal sequence portion, uracil containing nucleotides aresubstituted for thymine containing nucleotides for all but the last10-20 nucleotides of the universal sequence portion, and a polymerasethat will not accept or process uracil-containing templates is used foramplification of the nucleic acid, resulting in significant improvementof key sequencing metrics, FIG. 16A, FIG. 21, and FIG. 22. Furthermore,a blocking oligonucleotide comprising uracil containing nucleotides anda blocked 3′ end (e.g. 3′ddCTP) are used to promote priming of thenucleic acid by the random N-mer sequence and prevent preferentialbinding to portions of the nucleic acid that are complementary to theRead 1 Primer site, 1402. Additionally, product lengths are furtherlimited by inclusion of a small percentage of terminating nucleotides(e.g., 0.1-2% acyclonucleotides (acyNTPs)) (FIG. 16B) to reduceundesired amplification products.

An example of partial hairpin formation to prevent amplification ofundesired products is provided here. First, initial denaturation isachieved at 98° C. for 2 minutes followed by priming a random portion ofthe genomic DNA sequence by the random N-mer sequence acting as a primerfor 30 seconds at 4° C. (FIG. 15A). Subsequently, sequence extensionfollows as the temperature ramps at 0.1° C./second to 45° C. (held for 1second) (FIG. 15A). Extension continues at elevated temperatures (20seconds at 70° C.), continuing to displace upstream strands and creatinga first phase of redundancy (FIG. 15B). Denaturation occurs at 98° C.for 30 seconds to release genomic DNA for additional priming. After thefirst cycle, amplification products have a single 5′ tag (FIG. 15C).These aforementioned steps are repeated up to 20 times, for example bybeginning cycle 2 at 4° C. and using the random N-mer sequence to againprime the genomic DNA where the black sequence indicates portions of theadded 5′ tags (added in cycle 1) that cannot be copied (FIG. 15D).Denaturation occurs at 98° C. to again release genomic DNA and theamplification product from the first cycle for additional priming. Aftera second round of thermocycling, both 5′ tagged products and 3′ & 5′tagged products exist (FIG. 15E). Partial hairpin structures form fromthe 3′ & 5′ tagged products preventing amplification of undesiredproducts (FIG. 15F). A new random priming of the genomic DNA sequencebegins again at 4° C. (FIG. 15G).

Example 13: Adding Additional Sequences by Amplification

For the completion of sequencer-ready libraries, an additionalamplification (e.g., polymerase chain reaction (PCR) step) is completedto add additional sequences, FIG. 14C. In order to out-compete hairpinformation, a primer containing locked nucleic acid (LNAs) or lockednucleic acid nucleotides, is used. Furthermore, in cases where theinclusion of uracil containing nucleotides is used in a previous step, apolymerase that does not discriminate against template uracil containingnucleotides is used for this step. The results presented in FIG. 17 showthat a blocking oligonucleotide reduces start site bias, as measured bysequencing on an Illumina MiSeq sequencer. The nucleic acid template inthis case is yeast gDNA.

Example 14: Digital Processor

A conceptual schematic for an example control assembly, 1801, is shownin FIG. 18. A computer, 1802, serves as the central hub for controlassembly, 1801. Computer, 1802, is in communication with a display,1803, one or more input devices (e.g., a mouse, keyboard, camera, etc.)1804, and optionally a printer, 1805. Control assembly, 1801, via itscomputer, 1802, is in communication with one or more devices: optionallya sample pre-processing unit, 1806, one or more sample processing units(such as a sequence, thermocycler, or microfluidic device) 1807, andoptionally a detector, 1808. The control assembly may be networked, forexample, via an Ethernet connection. A user may provide inputs (e.g.,the parameters necessary for a desired set of nucleic acid amplificationreactions or flow rates for a microfluidic device) into computer, 1802,using an input device, 1804. The inputs are interpreted by computer,1802, to generate instructions. The computer, 1802, communicates suchinstructions to the optional sample pre-processing unit, 1806, the oneor more sample processing units, 1807, and/or the optional detector,1808, for execution. Moreover, during operation of the optional samplepre-processing unit, 1806, one or more sample processing units, 1807,and/or the optional detector, 1808, each device may communicate signalsback to computer, 1802. Such signals may be interpreted and used bycomputer, 1802, to determine if any of the devices require furtherinstruction. Computer, 1802, may also modulate sample pre-processingunit, 1806, such that the components of a sample are mixed appropriatelyand fed, at a desired or otherwise predetermined rate, into the sampleprocessing unit (such as the microfluidic device), 1807. Computer, 1802,may also communicate with detector, 1808, such that the detectorperforms measurements at desired or otherwise predetermined time pointsor at time points determined from feedback received from pre-processingunit, 1806, or sample processing unit, 1807. Detector, 1808, may alsocommunicate raw data obtained during measurements back to computer,1802, for further analysis and interpretation. Analysis may besummarized in formats useful to an end user via display, 1803, and/orprintouts generated by printer, 1805. Instructions or programs used tocontrol the sample pre-processing unit, 1806, the sample processingunit, 1807, and/or detector, 1808; data acquired by executing any of themethods described herein; or data analyzed and/or interpreted may betransmitted to or received from one or more remote computers, 1809, viaa network, 1810, which, for example, could be the Internet.

Example 15: Combinatorial Technique Via Ligation

As shown in FIG. 23A, beads 2301 are generated and covalently linked(e.g., via an acrydite moiety) to a partial P5 sequence 2302.Separately, in 50 μL, of each well of 4 96 well plates, anoligonucleotide 2303, comprising the remaining P5 sequence and a uniquepartial barcode sequence (indicated by bases “DDDDDD” in oligonucleotide2303), is hybridized to an oligonucleotide 2304 that comprises thereverse complement to oligonucleotide 2303 and additional bases thatoverhang each end of oligonucleotide 2303. Splint 2306 is generated.Each overhang is blocked (indicated with an “X” in FIG. 23) with 3′ C3Spacer, 3′ Inverted dT, or dideoxy-C(ddC) to prevent side productformation.

As shown in FIG. 23B, splints 2306 are each added to 4 96 deep wellplates, with each well comprising 2 mL beads 2301 and a splintcomprising a unique partial barcode sequence. In each well, the splint2306 hybridizes with the partial P5 sequence 2302 of beads 2301, via thecorresponding overhang of oligonucleotide 2304. Following hybridization,partial P5 sequence 2302 is ligated to oligonucleotide 2303 (which willtypically have been 5′ phosphorylated) via the action of a ligase, e.g.,a T4 ligase, at 16° C. for 1 hour. Following ligation, the products arepooled and the beads washed to remove unligated oligonucleotides.

As shown in FIG. 23C, the washed products are then redistributed intowells of 4 new 96 well plates, with each well of the plate comprising 2mL of beads 2301 and an oligonucleotide 2305 that has a unique partialbarcode sequence (indicated by “DDDDDD” in oligonucleotide 2305) and anadjacent short sequence (e.g., “CC” adjacent to the partial barcodesequence and at the terminus of oligonucleotide 2305) complementary tothe remaining overhang of oligonucleotide 2304. Oligonucleotide 2305also comprises a random N-mer (indicated by NNNNN″ in oligonucleotide2305). Via the adjacent short sequence, oligonucleotide 2305 ishybridized with oligonucleotide 2304 via the remaining overhang ofoligonucleotide 2304. Oligonucleotide 2305 is then ligated tooligonucleotide 2303 via the action of a ligase at 16° C. for 1 hour.Ligation of oligonucleotide 2305 to oligonucleotide 2303 results in thegeneration of a full barcode sequence. As shown in FIG. 23D, theproducts are then pooled, the oligonucleotide 2304 is denatured from theproducts, and the unbound oligonucleotides are then washed away.Following washing, a diverse library of barcoded beads is obtained, witheach bead bound to an oligonucleotide comprising a P5 sequence, a fullbarcode sequence, and a random N-mer. The generated library comprisesapproximately 147,000 different barcode sequences.

Example 16: Substitution of Uracil Containing Nucleotides for ThymineContaining Nucleotides in Barcode Primers

As shown in FIG. 33A, two barcode primers 3301 and 3302 suitable forPHASE amplification were used to amplify sample nucleic acid obtainedfrom a yeast genome. Following PHASE amplification, additional sequenceswere added (e.g., via bulk PCR) to generate sequencer-ready products.Barcode primers 3301 (also shown as U.2 in FIG. 33A) and 3302 (alsoshown at U.1 in FIG. 33A) comprised an identical sequence except thatbarcode primer 3301 comprised an additional uracil containingnucleotide-for-thymine containing nucleotide substitution at position3306. Sets of amplification experiments were run for each barcodeprimer, with each set corresponding to a particular blockeroligonucleotide mixed with the respective barcode primer at variousstoichiometries. For barcode primer 3302, sets of amplificationexperiments corresponding to a standard blocker oligonucleotide 3303, afull blocker oligonucleotide comprising bridged nucleic acid (BNAs) 3304(also shown as BNA blocker in FIG. 33A), or a full blockeroligonucleotide 3305 were conducted. Blocker oligonucleotides 3303 and3305 comprised uracil containing nucleotide-for-thymine containingnucleotide substitutions at all thymine containing nucleotide positionsand a ddC blocked end. In each set, the blocker oligonucleotide:barcodeprimer stoichiometry was either 0, 0.4, 0.8, or 1.2. For barcode primer3301, each type of blocker oligonucleotide 3303, 3304, and 3305 wastested at a 0.8 blocker oligonucleotide:barcode primer stoichiometry.

The size results of PHASE amplification products are depicted in FIG.33B. As shown, barcode primer 3302 (e.g., comprising the extra uracilcontaining nucleotide-for-thymine containing nucleotide substitution)coupled to blocker oligonucleotide 3303 generally produced the smallestamplification products across the stoichiometries tested. Results forbarcode primer 3302 with respect to blocker oligonucleotides 3304 and3305 varied, with sizes generally larger than results for blockeroligonucleotide 3303. For barcode primer 3301, amplification productsizes were also generally larger than those obtained for barcode primer3301 coupled to blocker oligonucleotide 3303 across the blockeroligonucleotides tested. The size results of sequencer-ready productsare depicted in FIG. 33C.

Key sequencing metrics obtained from the amplification products aredepicted in FIG. 33D. As shown, the fraction of unmapped reads (panel Iin FIG. 33D) was generally lower for sequencing runs for amplificationproducts generated from barcode primer 3302. For example, the fractionof unmapped reads for amplification products generated from barcodeprimer 3302 and blocker oligonucleotide 3303 at 0.8 blockeroligonucleotide:barcode primer stoichiometry was approximately 7-8%,whereas results obtained using barcode primer 3301 at the sameconditions was approximately 17-18%. Moreover, Q40 error rates (panel IIin FIG. 33D) were also lower for barcode primer 3302. For example, Q40error rate for amplification products generated from barcode primer 3302and blocker oligonucleotide 3303 at 0.8 blocker oligonucleotide:barcodeprimer stoichiometry was approximately 0.105%, whereas results obtainedusing barcode primer 3301 at the same conditions was approximately0.142%. Read 1 start site (panel III) and Read 2 start site (panel IV)relative entropies determined during sequencing are shown in FIG. 33E.

Example 17: Post-Synthesis Functionalization of Gel Beads Via DisulfideExchange

Gel beads comprising disulfide bonds were generated according to one ormore methods described herein. The gel beads were then reacted with TCEPat ratios of molecules of TCEP to gel beads (TCEP:GB). The tested ratioswere 0, 2.5 billion, and 10.0 billion. The TCEP functions as a reducingagent to generate free thiols within the gel beads. Following reduction,the gel beads were washed once to remove the TCEP. Next, the generatedfree thiols of the gel beads were reacted with an acrydite-S—S-P5species (e.g., 3505 in FIG. 35A) to link the acrydite-S—S-P5 to the gelbeads via Michael addition chemistry as shown in FIG. 35A. Differentratios of acrydite-S—S-P5 to each type (e.g., ratio of TCEP:GB used togenerate free thiols on the gel beads) of the activated gel beads weretested. The tested ratios of acrydite-S—S-P5 species to activated gelbeads (P5:GB) were 50 million, 500 million, and 5 billion.

Following syntheses, the gel beads from each reaction were washed andtreated with DTT in a reaction mixture to degrade the gel beads andrelease any bound acrydite-S—S-P5 species. An aliquot of each reactionmixture was entered into a lane of a gel and free oligonucleotidessubject to gel electrophoresis as shown in FIG. 36 (e.g., lanes 3-11 inFIG. 36). A 50 picomole acrydite-S—S-P5 standard was also run (e.g.,lane 1 in FIG. 36) along with a 25 base pair ladder (e.g., lane 2 inFIG. 36). Bands corresponding to loaded acrydite-S—S-P5 were generatedin lanes 5 and 8 (indicated by arrows in FIG. 36). Lane 5 corresponds togel beads treated at a TCEP:GB ratio of 2.5 billion and the TCEP treatedgel beads reacted with acrydite-S—S-P5 at a P5:GB ratio of 5 billion.Lane 8 corresponds to gel beads treated at a TCEP:GB ratio of 10.0billion and the TCEP treated gel beads reacted with acrydite-S—S-P5 at aP5:GB ratio of 5 billion.

Example 18: Post-Synthesis Functionalization of Gel Beads Via DisulfideExchange

Gel beads comprising disulfide bonds were generated according to one ormore methods described herein. The gel beads were then reacted with TCEPin 0.1M phosphate buffer at a concentration of 4 μg TCEP/100,000 gelbeads. The TCEP can function as a reducing agent to generate gel beadswith free thiol groups. Following reduction, the gel beads were washedonce to separate the gel beads from the TCEP. Next, the free thiols ofthe gel beads were reacted with 2,2′-dithiopyridine (e.g., 3507 in FIG.35B) in a saturated solution (−0.2 mM) of 2,2′-dithiopyridine to linkpyridine groups to the gel beads via disulfide exchange chemistry asshown in FIG. 35B. Following synthesis, the gel beads were washed threetimes to remove excess 2,2′-dithiopyridine.

The washed gel beads were then reacted with an oligonucleotide 3702comprising a full construct barcode (FCBC—e.g., an oligonucleotidecomprising P5, a barcode sequence, R1, and a random N-mer) sequence atone end and a free thiol group at its other end. Two reactions werecompleted at two different ratios of molecules of FCBC to gel beads(e.g., FCBC:GB) and the reactions were allowed to proceed overnight. Thetested FCBC:GB ratios were 400 million and 1.6 billion. Oligonucleotide3702 was initially supplied with its free thiol group protected in adisulfide bond, shown as 3701 in FIG. 37A. To generate the free thiol asin oligonucleotide 3702, oligonucleotide 3701 was treated with 0.1 M DTTin 1× Tris-EDTA buffer (TE) buffer for 30 minutes. Salt exchange on aSephadex (NAP-5) column was used to remove DTT after reduction andpurify oligonucleotide 3702. For each reaction, purifiedoligonucleotides 3702 were then reacted with the dithio-pyridine speciesof the gel beads via thiol-disulfide exchange (e.g., see FIG. 35B) togenerate gel beads comprising oligonucleotide 3702. Following thereaction, the gel beads were purified by washing the beads three times.

For comparison purposes, gel beads comprising disulfide bonds and theFCBC sequence were also generated via polymerization of monomers asdescribed elsewhere herein. The FCBC was linked to a monomer comprisingan acrydite species that was capable of participating in apolymerization with acrylamide and bis(acryloyl)cystamine to generatethe gel beads. The FCBC sequence was linked to the gel beads via theacrydite moiety.

Following syntheses, the gel beads from each reaction were washed andtreated with DTT in a reaction mixture to degrade the gel beads andrelease any bound oligonucleotide 3702. Gel beads comprising the FCBCsequence that were synthesized via polymerization were also treated withDTT in a reaction mixture. An aliquot of each reaction mixture wasentered into a lane of a gel and free oligonucleotides subject to gelelectrophoresis as shown in FIG. 37B. As shown in the gel photographdepicted in FIG. 37B, lane 1 corresponds to a 50 base pair ladder; lane2 corresponds to gel beads functionalized via disulfide exchangechemistry at an FCBC:GB ratio of 400 million; lane 3 corresponds to gelbeads functionalized via disulfide exchange chemistry at an FCBC:GBratio of 1.6 billion; and lane 4 corresponds to functionalized gel beadsgenerated via polymerization of acrydite species. Bands corresponding toloaded oligonucleotides were generated for functionalized gel beadsgenerated at both FCBC:GB ratios and were at a similar position to theband generated for functionalized gel beads generated via polymerizationof acrydite species.

Following syntheses, gel beads from each reaction were also washed andstained with SYBR Gold fluorescent stain. Gel beads comprising the FCBCsequence that were synthesized via polymerization were also stained withSYBR Gold. SYBR Gold can stain functionalized beads by intercalating anybound oligonucleotides. Following staining, the beads were pooled andimaged using fluorescence microscopy, as shown in the micrographdepicted in FIG. 37C. Brighter beads (3704) in FIG. 37C correspond tobeads functionalized during polymerization of the beads and dim beads(still showing SYBR gold signal) (3705) correspond to beadsfunctionalized with disulfide exchange chemistry after gel beadgeneration. Loading of oligonucleotides via disulfide-exchange wasapproximately 30% of that achieved with functionalization of beadsduring gel bead polymerization.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1-27. (canceled)
 28. A method, comprising: (a) partitioning a firstplurality of beads and a first plurality of nucleic acid moleculescomprising a first partial barcode sequence (B1) into a first pluralityof wells, wherein said first plurality of wells comprises M wells,wherein each well of said M wells comprises nucleic acid moleculescomprising a different first partial barcode sequence, and wherein saidfirst plurality of nucleic acid molecules comprises M different firstpartial barcode sequences m nucleotides in length; (b) attaching nucleicacid molecules of said first plurality of nucleic acid molecules tobeads of said first plurality of beads to yield a second plurality ofbeads comprising M different first partial barcode sequences attachedthereto; (c) pooling said second plurality of beads; (d) partitioningsaid second plurality of beads and a second plurality of nucleic acidmolecules comprising a second partial barcode sequence (B2) into asecond plurality of wells, wherein said second plurality of wellscomprises N wells, wherein each well of said N wells comprises adifferent second partial barcode sequence, and wherein said secondplurality of nucleic acid molecules comprises N different second partialbarcode sequences n nucleotides in length; and (e) attaching nucleicacid molecules of said second plurality of nucleic acid molecules tobeads of said second plurality of beads to provide a third plurality ofbeads comprising N×M different barcode sequences attached thereto,wherein each barcode sequence of said N×M different barcode sequencescomprises a first partial barcode sequence (B1) and a second partialbarcode sequence (B2); and wherein M≥96, N≥96, m≥4, and n≥4.
 29. Themethod of claim 28, wherein M≥384 and N≥384.
 30. The method of claim 28,wherein said first plurality of beads comprises, covalently attachedthereto, a plurality of nucleic acid anchor molecules comprising a firstadapter sequence, wherein said first plurality of nucleic acid moleculescomprises a sequence complementary to said first adapter sequence, andwherein (b) comprises hybridizing said first plurality of nucleic acidmolecules to said first adapter sequence.
 31. The method of claim 30,wherein (b) further comprises ligating said first plurality of nucleicacid molecules to said anchor molecules to generate said secondplurality of beads.
 32. The method of claim 31, wherein said firstplurality of nucleic acid molecules comprises a second adapter sequence,wherein said second plurality of nucleic acid molecules comprises asequence complementary to said second adapter sequence, and wherein (e)comprises hybridizing said second plurality of nucleic acid molecules tosaid second adapter sequence.
 33. The method of claim 32, wherein (e)further comprises ligating said second plurality of nucleic acidmolecules to said second adapter sequence to generate said thirdplurality of beads.
 34. The method of claim 33, wherein said secondplurality of nucleic acid molecules comprises a third adapter sequencecomprising a poly-T sequence, a random sequence, or a targeted primersequence.
 35. The method of claim 30, wherein said anchor molecules aresingle-stranded.
 36. The method of claim 30, wherein said first adaptersequence is single-stranded, wherein said first plurality of nucleicacid molecules are partially double-stranded molecules comprising (i) afirst single-stranded overhang complementary to said first adaptersequence and (ii) a second single-stranded overhang, and wherein (b)comprises hybridizing said first single-stranded overhang to said firstadapter sequence and ligating said first plurality of nucleic acidmolecules to said anchor molecules to generate a plurality of partialbarcode molecules comprising said first adapter sequence and said B1sequence.
 37. The method of claim 36, wherein said second plurality ofnucleic acid molecules are partially double-stranded moleculescomprising a third single-stranded overhang complementary to said secondsingle-stranded overhang, and wherein (e) comprises hybridizing saidsecond single-stranded overhang to said third single-stranded overhangand ligating said second plurality of nucleic acid molecules to saidplurality of partial barcode molecules.
 38. The method of claim 37,wherein said second plurality of nucleic acid molecules comprises acapture sequence comprising a poly-T sequence, a random sequence, or atargeted primer sequence.
 39. The method of claim 28, wherein said firstpartial barcode sequence and said second partial barcode sequence areseparated by a linking sequence (L) that is at least 1 nucleotide inlength.
 40. The method of claim 39, wherein each bead of said thirdplurality of beads comprises a barcode molecule having the structureB1-L-B2-C, wherein C is a capture sequence configured to hybridize to anucleic acid molecule.
 41. The method of claim 40, wherein said Ccomprises a poly-T sequence, a random sequence, or a targeted primersequence.
 42. The method of claim 40, wherein each bead of said thirdplurality of beads comprises a barcode molecule having the structureA-B1-L-B2-C, wherein A is an adapter sequence, and wherein A is the sameacross said third plurality of beads.
 43. The method of claim 42,wherein said adapter sequence comprises a primer sequence or a primerbinding sequence.
 44. The method of claim 43, wherein said primersequence or said primer binding sequence is a sequencing primer sequenceor a sequencing primer binding sequence.
 45. The method of claim 42,wherein said adapter sequence comprises a sequence configured to attachto a flow cell of a sequencer.
 46. The method of claim 42, wherein saidbarcode molecule is single-stranded.
 47. The method of claim 28, whereinsaid first plurality of beads is a plurality of gel beads.
 48. Themethod of claim 47, wherein gel beads of said plurality of gel beads aredegradable upon application of a stimulus.
 49. The method of claim 48,wherein said stimulus is a thermal stimulus, a chemical stimulus, abiological stimulus, or a photo-stimulus.
 50. The method of claim 48,wherein said stimulus is a reducing agent.
 51. The method of claim 28,wherein nucleic acid molecules of said first plurality of nucleic acidmolecules are releasable from said bead upon application of a stimulus.52. The method of claim 51, wherein said stimulus is a thermal stimulus,a chemical stimulus, a biological stimulus, or a photo-stimulus.
 53. Themethod of claim 51, wherein said stimulus is a reducing agent.
 54. Themethod of claim 28, further comprising, subsequent to (e), (f) poolingsaid third plurality of beads; (g) partitioning said third plurality ofbeads and a third plurality of nucleic acid molecules comprising a thirdpartial barcode sequence (B3) into a third plurality of wells, whereinsaid third plurality of wells comprises Z wells, wherein each well ofsaid Z wells comprises a different third partial barcode sequence, andwherein said third plurality of nucleic acid molecules comprise Zdifferent third partial barcode sequences z nucleotides in length; and(h) attaching nucleic acid molecules of said third plurality of nucleicacid molecules to beads of said third plurality of beads to provide afourth plurality of beads comprising N×M×Z different barcode sequencesattached thereto, wherein each barcode sequence of said N×M×Z differentbarcode sequences comprises a first partial barcode sequence (B1), asecond partial barcode sequence (B2), and a third partial barcodesequence (B3); and wherein Z≥96 and z≥6.
 55. The method of claim 54,wherein said first partial barcode sequence segment and said secondpartial barcode sequence are separated by a linking sequence (L1) atleast 1 nucleotide in length, and wherein said second partial barcodesequence and said third partial barcode sequence are separated by alinking sequence (L2) at least 1 nucleotide in length.
 56. The method ofclaim 55, wherein L1 and L2 are different sequences.
 57. The method ofclaim 55, wherein each bead of said fourth plurality of beads comprisesa barcode molecule having the structure A-B1-L1-B2-L2-B3-C, wherein A isan adapter sequence, and wherein C is a capture sequence configured tohybridize to a nucleic acid molecule.